CN107557478A - A kind of method and its application that amplification the Changjiang river fish chondriogen total order is combined based on degenerate primer - Google Patents
A kind of method and its application that amplification the Changjiang river fish chondriogen total order is combined based on degenerate primer Download PDFInfo
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Abstract
The present invention provides a kind of method and its application that amplification the Changjiang river fish chondriogen total order is combined based on degenerate primer, the primer combination is compared by the mitochondria total order of Acipenseridae, Salangidae, Cyprinidae, salmonidae, Anguillidae, catfish section, Chang sections, Medaka section, Zhen sections, conjunction gill Gyrinocheilidae, Sushi sections, Acenthogobius flavimanus section, murrel section, Catfish sections, Cobitidae fish, designed in conservative region, including Mt 1F, Mt 1R;Mt‑2F,Mt‑2R;Mt‑3F,Mt‑3R;Mt‑4F,Mt‑4R;Mt‑5F,Mt‑5R;Mt 6F, Mt 6R and Mt 7F, Mt 7R.The present invention enters performing PCR amplification corresponding gene fragment using above-mentioned 7 pairs of primers, and chondriogen complete sequence is obtained by connection conversion, sequencing splicing, and primer combined few, expanding effect is good, effectively can carry out taxonomic identification to monitoring fish, fish-egg.The present invention can obtain the chondriogen total order of a variety of fingerlings, versatile, obtained data may be used in fish mitochondrial genomes research, improve mitochondria database based on fish mitochondria conservative region design degenerate primer.
Description
Technical field
The present invention relates to biology field, refers specifically to a kind of based on degenerate primer combination amplification the Changjiang river fish mitochondria
The method and its application of gene total order
Background technology
Traditional fish species classification tends to rely on its formalness feature, including body length, body height, body colour, fin ray number,
Relative measurement of speckle or body parts etc..But some fish distribution scopes are wide, species is more, and in different growth steps
Section morphological feature changes greatly, and this carries out fish species identification to researcher and causes certain difficulty.In addition, some fish
It is similar in early development stage formalness, some deviations often occur in Identification of Species, even if professional researcher
Unavoidable error.At present, many ecological monitoring study, for example the cultivation effect of artificial fish nest is studied, it is necessary to which fish nest will be adhered to
On fish-egg peel off, could carry out species identification in indoor culture to the stage for having obvious properties and characteristicses, spend the time longer, and
Because mortality is larger, causing final fish nest cultivation effect to be evaluated has deviation.So one kind can quickly, accurately carry out fish
The technology of kind classification is very necessary.
MtDNA of fishes (mitochondrial DNA) have molecule is small, simple in construction, evolutionary rate is fast, different zones evolutionary rate is deposited
It is a relatively independent replicator, just because of these features, fish chondriogen has become the difference the features such as
The research meanses that fish systematic growth and population genetic are commonly used, it is increasing in recent years ecological with the popularization of sequencing technologies
Monitoring is had using analysis chondriogen come the species composition in survey region, conventional fish chondriogen fragment
16srDNA, COI, Cytb1, Cytb4, D-loop, Liu Qigen etc. expand to said gene fragment, obtain Thousand-Island Lake storehouse
The Fish Community Structures in area, it is consistent with monitoring and surveying result over the years.But because above-mentioned fragment is shorter, distinguishing some genetic distances
Error occurs in more similar fingerling often, so, the result being compared using mitochondria total order is the most reliable.At present,
Ncbi database shares 7539 fish Mitochondrial gene sequences, wherein, the fish Mitochondrial gene sequence that Chinese Regional uploads
2271 are shared, mostlys come from Dongting Lake, Poyang Lake, Xianghe, Lijiang River, the Yangtze river basin and each province and city, whole database includes
The fish Mitochondrial gene sequence of most of Yangtze river basin, laid the foundation for the fingerling identification in later stage of the invention.But at present, often
MtDNA amplification meanses have 2 kinds, and one kind is to obtain total order based on nearly source species design primer, amplification；Another kind is based on high pass
Measure sequence and carry out assembling acquisition.First method excessively relies on nearly source species, and second of cost is too high.So Yi Zhongqiang
Universality, economically viable fingerling mitochondria total order assay method is very necessary.The present invention is by continuously attempting to, by Yangtze River Water
Rich a few section fish in system：Acipenseridae, Salangidae, Cyprinidae, salmonidae, Anguillidae, Cyprinidae, catfish section, Chang sections, Medaka Ke, Zhen sections, the conjunction gill
Gyrinocheilidae, Sushi Ke, Acenthogobius flavimanus section, murrel Ke, Catfish sections, the mitochondria total order of Cobitidae fish are compared, and degeneracy is designed in conservative region
Primer, 7 pairs of primers are obtained by total length 16000bp or so sequences segmentation into 7 fragments, the later stage expanded by PCR, is connected and is turned
Change, sequencing splicing obtain the complete sequence of chondriogen, improve the accuracy of fingerling identification, while the extensive use of this method
Also fingerling Mitochondrial gene sequence database in the Yangtze river basin in NCBI preferably will be improved, except the application in being identified in fingerling,
This method also has very important application value in the research of fish population evolution, species diversity and genetic diversity.
The content of the invention
It is an object of the invention to provide a kind of side that amplification the Changjiang river fish chondriogen total order is combined based on degenerate primer
Method and its application
In order to realize the object of the invention, a kind of degenerate primer group for being used to expand fish chondriogen total order of the invention
Close, including：
Pair of primers：
Mt-1F:5’-ARWTTTACCGGCCCTHTTA-3’
Mt-1R:5’-AGATAGAAACTGACCTGGATT-3’
Second pair of primer：
Mt-2F:5’-GTTCAACGATTAAAGTCCTAC-3’
Mt-2R:5’-AARRGTAGRTGRATGTCGCTG-3’
3rd pair of primer：
Mt-3F:5’-GMAGGCCTYGATCCTRCAAA-3’
Mt-3R:5’-GCYGCRTCTTGRAAKCCTA-3’
4th pair of primer：
Mt-4F:5’-GTTTCAAGYCARYCRCATAA-3’
Mt-4R:5’-TRAGCCGAAATCARAKGTC-3’
5th pair of primer：
Mt-5F:5’-AGTAYRWGTGACTTCCAATCA-3’
Mt-5R:5’-CATAGCTKYYRCTTGGAKTTG-3’
6th pair of primer：
Mt-6F:5’-TAGGAACCAAAAACTCTTGGT-3’
Mt-6R:5’-ACAACGGTGGTTYTTCAAG-3’
7th pair of primer：
Mt-7F:5’-TGACTTGAARAACCACCGT-3’
Mt-7R:5’-ARWTTTACCGGCCCTHTTA-3
Wherein：Y represents base C or T；M represents base C or A；R represents base A or G；W represents base A or T；K represents alkali
Base T or G；H represents base A, T or C.
The present invention also provides the Tiangeng DNA reagents for being used to expand fish mitochondria homologous segment of above-mentioned degenerate primer combination
Box and reagent.The reagent includes one or more LA Taq archaeal dna polymerases, PCR reaction buffers, dNTPs.
The present invention also provides the degenerate primer and combines the application in fingerling classification is carried out, and comprises the following steps：
1) DNA in sample is extracted；
2) using the DNA of extraction in step 1) as template, pcr amplification reaction is carried out respectively using above-mentioned 7 pairs of primers；
3) 7 genetic fragments for obtaining step 2) carry out gel extraction, connection conversion, sequencing splicing, obtain mitochondria
Gene complete sequence；
4) sequence obtained in NCBI to step 3) carries out Blast comparisons, determines species.
Preferably, PCR reaction systems are in every 25 μ l systems：10xbuffer 2.5 μ l, MgCl2(25mM) 1.5 μ l,
The μ l of dNTPs (10mM) 1, concentration be 100pmol/ μ l each 0.5 μ l of positive anti-primer, the μ l of template DNA 1, Taq enzyme (5U/ μ l) 0.25
μ l, ddH2O 17.75μl。
Preferably, PCR reaction conditions are 94 DEG C of pre-degeneration 3min, and 94 DEG C of denaturation 30s, 48-65 DEG C of annealing 30s, 72 DEG C are prolonged
Stretch 2min, 30 circulations, 72 DEG C of extension 10min.Wherein, preferably 55 DEG C of annealing temperature.
Preferably, linked system is in every 5 μ l junctors system：Glue reclaim product 1-4 μ l, pEASY-T1 Cloning
Vector 1 μ l, ddH2O 3μl；Coupled reaction system is：25 DEG C of connection 20-30min；4 DEG C preserve for transformed clone sequencing.
Wherein, linked system glue reclaim product preferably 1 μ l, preferably 25 DEG C connection 25min of reaction system.
Specifically, the present invention utilizes several representative Acipenseridaes, Salangidae, Cyprinidae, salmonidae, Anguillidae, Cyprinidae, catfish
Section, Chang sections, Medaka Ke, Zhen sections, conjunction gill Gyrinocheilidae, Sushi Ke, Acenthogobius flavimanus section, murrel Ke, Catfish sections, the chondriogen total order of Cobitidae fish are entered
Row is compared, and degenerate primer is designed according to conservative region, recycles degenerate primer to expand known and species to be measured fish mitochondria
Gene is verified that on the one hand the accuracy of primer designed by checking, on the other hand verifies whether primer combination has 100%
Amplification efficiency, each pair primer can preferably amplify respective segments.Flow is as follows：
1) universal primer designs
From NCBI websites download 2 kinds of Acipenseridae, 2 kinds of Salangidae, 2 kinds of salmonidae, 2 kinds of Anguillidae, 9 kinds of Cyprinidae, a kind of catfish section,
2 kinds of Chang sections, a kind of Zhong , Zhen sections of Medaka section 1, close 1 Zhong , Sushi sections of gill Gyrinocheilidae 2, a kind of Zhong , Acenthogobius flavimanus section, 2 kinds of Zhong , Catfish sections of murrel section 1, Cobitidae 4
The Mitochondrial gene sequence of kind fish：Acipenser sinensis(KJ174513.1),Acipenser dabryanus
(AY510085.1),Protosalanx hyalocranius(NC_024109.1),Hemisalanx brachyrostralis
(NC_029157.1),Hucho bleekeri(HM804473.1),Hucho taimen(KJ711550.1),Anguilla
japonica(CM002536.1),Anguilla marmorata(NC_006540.1),Aphyocypris chinensis
(NC_008650.1),Ctenopharynodon idellus(NC_025590.1),Cyprinus carpio
(KU050703.1), Xenocypris davidi (KF039718.1), Rhodeus ocellatus (DQ026430.1),
Ancherythroculter nigrocauda(NC_021414.1),Coreius heterodon(NC_020042.1),
Onychostoma sima(NC_021972.1),Lepturichthys fimbriata(NC_024609.1),Silurus
asotus(JX256247.1),Pelteobagrus vachelli(HM746660.1),Mystus macropterus(NC_
019592.1),Oryzias latipes(NC_004387.1),Tylosurus melanotus(KU605633.1),
Monopterus albus(KP779625.1),Coreosiniperca roulei(NC_024432.1),Siniperca
chuatsi(NC_015822.1),Rhinogobius giurinus(KU871066.1),Channa argus
(KC823605.1),Tylosurus melanotus(KU605633.1),Takifugu obscurus(EF672695.1),
Leptobotia elongata(JQ230103.1),Misgurnus anguillicaudatus(KM186181.1),
Sinibotia superciliaris(JX683724.1),Triplophysa pappenheimi(NC_033972.1).Utilize
33 kinds of sequences are compared DNAMAN softwares, according to conservative region, design 7 pairs of degenerate primers (SeqIDNo.1-14)：
Pair of primers (amplification length is about 2500bp):
Mt-1F:5’-ARWTTTACCGGCCCTHTTA-3’
Mt-1R:5’-AGATAGAAACTGACCTGGATT-3’
Second pair of primer (amplification length is about 2700bp):
Mt-2F:5’-GTTCAACGATTAAAGTCCTAC-3’
Mt-2R:5’-AARRGTAGRTGRATGTCGCTG-3’
3rd pair of primer (amplification length is about 2000bp):
Mt-3F:5’-GMAGGCCTYGATCCTRCAAA-3’
Mt-3R:5’-GCYGCRTCTTGRAAKCCTA-3’
4th pair of primer (amplification length is about 2900bp):
Mt-4F:5’-GTTTCAAGYCARYCRCATAA-3’
Mt-4R:5’-TRAGCCGAAATCARAKGTC-3’
5th pair of primer (amplification length is about 2300bp):
Mt-5F:5’-AGTAYRWGTGACTTCCAATCA-3’
Mt-5R:5’-CATAGCTKYYRCTTGGAKTTG-3’
6th pair of primer (amplification length is about 2400bp):
Mt-6F:5’-TAGGAACCAAAAACTCTTGGT-3’
Mt-6R:5’-ACAACGGTGGTTYTTCAAG-3’
7th pair of primer (amplification length is about 2500bp):
Mt-7F:5’-TGACTTGAARAACCACCGT-3’
Mt-7R:5’-ARWTTTACCGGCCCTHTTA-3’
Wherein：Y represents base C or T；M represents base C or A；R represents base A or G；W represents base A or T；K represents alkali
Base T or G；H represents base A, T or C.Wherein, first pair of primer extension product is 12SRNA*, tRNA-Val, 16SRNA*, there is 2*
2*3=12 kind combining forms；Second pair of primer extension product is 16SRNA*, tRNA-Leu, ND1, tRNA-Ile, tRNA-Gln,
TRNA-Met, ND2, tRNA-Trp, tRNA-Ala, tRNA-Asn, tRNA-Cys*, there are 2*2*2=8 kind combining forms；3rd pair
Primer extension product is tRNA-Asn*, tRNA-Cys, tRNA-Tyr, COI, tRNA-Ser, tRNA-Asp, COII*, there is 2*2*
2*2*2*2*2=128 kind combining forms；4th pair of primer extension product is tRNA-Asp*, COII, tRNA-Lys, ATP8,
ATP6, COIII, tRNA-Gly, ND3, tRNA-Arg, ND4L, ND4*, there are 2*2*2*2*2*2*2=128 kind combining forms；The
Five pairs of primer extension products are ND3*, tRNA-Arg, ND4L, ND4, tRNA-His, tRNA-Ser*, there is 2*2*2*2*2*2*2*2
=256 kinds of combining forms；6th pair of primer extension product is tRNA-His*, tRNA-Ser, tRNA-Leu, ND5*, there is 2*2*2*
2*2*=32 kind combining forms；7th pair of primer extension product is ND5*, ND6, tRNA-Glu, CYTB, tRNA-Thr, tRNA-
Pro, D-loop, tRNA-Phe, 12SRNA*, there are 2*2*2*2*2*2*2*3=384 kind combining forms.
2) DNA in sample is extracted
Carried using TIANGENTIANampGenomicDNAKit (blood/cell/tissue extracting genome DNA) kit
Take species DNA to be checked.During extraction, first sample tissue (fin, muscle, fish-egg etc.) is shredded, a small amount of sample of picking buffers in 200 μ l
In liquid GA, 20 μ l Proteinase K Solutions are added, are mixed, digest 30-60min in 56 DEG C.200 μ l buffer solution GB are added, it is fully reverse
Mix, 70 DEG C of placement 10min.200 μ l absolute ethyl alcohols are added, all liq is transferred in adsorption column, 12000rpm centrifuges 2 points
Clock.Waste liquid is abandoned, 500 μ lGD are added into adsorption column, 2min is centrifuged in 12000rpm.Waste liquid is abandoned, 600 μ l drifts are added to adsorption column
Washing lotion PW, 2min is centrifuged in 12000rpm.Repeat this step.Then waste liquid is abandoned, 2min is centrifuged in 12000rpm, and it is thorough in room temperature
Dry at bottom.Adsorption column is placed in new EP pipes, 50 μ lddH are added to adsorption column2O, 2min is centrifuged in 12000rpm.It will obtain
DNA is placed in -20 degree and preserved.
3) PCR amplifications, recovery, conversion, clone, sequencing
Using above-mentioned 7 pairs of primers, performing PCR is entered to expand corresponding purpose fragment to the species DNA to be checked of extraction respectively.
PCR reaction systems are in every 25 μ l systems：10xbuffer2.5 μ l, MgCl2(25mM) 1.5 μ l, dNTPs (10mM) 1 μ l, concentration
For 100pmol/ μ l each 0.5 μ l of positive anti-primer, the μ l of template DNA 1, Taq enzyme (5U/ μ l) 0.25 μ l, ddH2O 17.75μl。PCR
Reaction condition is 94 DEG C of pre-degeneration 3min, and 94 DEG C of denaturation 30s, 48-65 DEG C of annealing 30s, 72 DEG C of extension 2min, 30 circulate, 72
DEG C extension 10min.Wherein, preferably 55 DEG C of annealing temperature.
PCR primer is identified into clip size and gel extraction with 1% agarose gel electrophoresis, connects, converts, sequencing.Even
Junctor system is in every 5 μ l systems：Glue reclaim product 1-4 μ l, pEASY-T1CloningVector1 μ l, ddH2O3μl；Connection is anti-
The condition is answered to be：25 DEG C of connection 20-30min；4 DEG C preserve for converting.Wherein, linked system glue reclaim product preferably 1 μ l, reaction
Preferably 25 DEG C connection 25min of system.
5 μ l connection product is transferred in 100 μ lDH5 α competent cells, places 30min on ice, then 42 DEG C of heat shocks
40s, 2min is placed on ice.The product of conversion is added in 800 μ lLB culture mediums, 90min is cultivated in 37 DEG C of 300rpm.Take
100 μ l nutrient solutions coated plate on the LBA culture mediums of the benzyl containing ammonia, 37 DEG C are incubated overnight, and carry out choosing bacterium culture sequencing.
4) sequence assembly, species are determined
Obtain 7 sections of sequences are spliced, obtain complete chondriogen total order, Blast is compared on NCBI, mirror
Determine species information.
7 pairs of degenerate primers of the invention by design, corresponding purpose fragment can be preferably amplified, can by splicing
The full sequence of fingerling chondriogen is obtained, taxonomic identification effectively can be carried out to fingerling to be measured using mitochondria total order.
The present invention has advantages below：
1. applicability：Traditional taxology requirement carries out Identification of Species by distinguishing the difference of fish body form, to identification
The specialty of personnel requires very high, is difficult to complete without the people of a large amount of fish Morphological Identification experiences.And the degenerate primer of the present invention, only
Want operating personnel that there is basic molecule experiments operational capacity can be competent at, it is less demanding to taxology professional knowledge, have more
High applicability.
2. high efficiency：Traditional form identification needs take a significant amount of time, especially when collecting fish-egg, it is necessary to which temporarily supporting can
The discriminating phase could carry out appraisal, waste time and energy.And the degenerate primer of the present invention, do not influenceed by the puberty, as long as being collected into
Material comprising fish body DNA information can start appraisal.
3. versatility：The present invention, can be to all the Changjiang river for the Changjiang river fish mitochondria conservative region design degenerate primer
Fish are effectively expanded.
4. accuracy：The degenerate primer that the present invention designs carries out Molecular Identification by chondriogen total order, greatly improves
The accuracy of Molecular Identification, and avoid and error is identified as caused by artificial origin.
Brief description of the drawings
Fig. 1 is 7 mitochondria bases of the 12 kinds of fish amplified in the embodiment of the present invention 2 using above-mentioned 7 pairs of degenerate primers
Because of fragment；Wherein, middle swimming lane is that DNA Marker, A~G are respectively amplified production corresponding to 7 pairs of degenerate primers, and 1~12 swims
Road is 12 samples (wherein, 3,4,11,12 be unknown fingerling, other for known fingerling).
Embodiment
Embodiment 1 expands the design of the degenerate primer combination of fish chondriogen
From NCBI websites download 2 kinds of Acipenseridae, 2 kinds of Salangidae, 2 kinds of salmonidae, 2 kinds of Anguillidae, 9 kinds of Cyprinidae, a kind of catfish section,
2 kinds of Chang sections, a kind of Zhong , Zhen sections of Medaka section 1, close 1 Zhong , Sushi sections of gill Gyrinocheilidae 2, a kind of Zhong , Acenthogobius flavimanus section, 2 kinds of Zhong , Catfish sections of murrel section 1,4 kinds of Cobitidae
The Mitochondrial gene sequence of fish：Acipenser sinensis(KJ174513.1),Acipenser dabryanus
(AY510085.1),Protosalanx hyalocranius(NC_024109.1),Hemisalanx brachyrostralis
(NC_029157.1),Hucho bleekeri(HM804473.1),Hucho taimen(KJ711550.1),Anguilla japonica
(CM002536.1),Anguilla marmorata(NC_006540.1),Aphyocypris chinensis(NC_008650.1),
Ctenopharynodon idellus (NC_025590.1), Cyprinus carpio (KU050703.1), Xenocypris davidi
(KF039718.1),Rhodeus ocellatus(DQ026430.1),Ancherythroculter nigrocauda(NC_
021414.1),Coreius heterodon(NC_020042.1),Onychostoma sima(NC_021972.1),Lepturichthys
fimbriata(NC_024609.1),Silurus asotus(JX256247.1),Pelteobagrus vachelli
(HM746660.1),Mystus macropterus(NC_019592.1),Oryzias latipes(NC_004387.1),
Tylosurus melanotus(KU605633.1),Monopterus albus(KP779625.1),Coreosiniperca roulei
(NC_024432.1),Siniperca chuatsi(NC_015822.1),Rhinogobius giurinus(KU871066.1),
Channa argus(KC823605.1),Tylosurus melanotus(KU605633.1),Takifugu obscurus
(EF672695.1),Leptobotia elongata(JQ230103.1),Misgurnus anguillicaudatus(KM186181.1),
Sinibotia superciliaris(JX683724.1),Triplophysa pappenheimi(NC_033972.1).Utilize DNAMAN
33 kinds of sequences are compared software, according to conservative region, design 7 pairs of degenerate primers (SeqIDNo.1-14)：
Pair of primers (amplification length is about 2500bp):
Mt-1F:5’-ARWTTTACCGGCCCTHTTA-3’
Mt-1R:5’-AGATAGAAACTGACCTGGATT-3’
Second pair of primer (amplification length is about 2700bp):
Mt-2F:5’-GTTCAACGATTAAAGTCCTAC-3’
Mt-2R:5’-AARRGTAGRTGRATGTCGCTG-3’
3rd pair of primer (amplification length is about 2000bp):
Mt-3F:5’-GMAGGCCTYGATCCTRCAAA-3’
Mt-3R:5’-GCYGCRTCTTGRAAKCCTA-3’
4th pair of primer (amplification length is about 2900bp):
Mt-4F:5’-GTTTCAAGYCARYCRCATAA-3’
Mt-4R:5’-TRAGCCGAAATCARAKGTC-3’
5th pair of primer (amplification length is about 2300bp):
Mt-5F:5’-AGTAYRWGTGACTTCCAATCA-3’
Mt-5R:5’-CATAGCTKYYRCTTGGAKTTG-3’
6th pair of primer (amplification length is about 2400bp):
Mt-6F:5’-TAGGAACCAAAAACTCTTGGT-3’
Mt-6R:5’-ACAACGGTGGTTYTTCAAG-3’
7th pair of primer (amplification length is about 2500bp):
Mt-7F:5’-TGACTTGAARAACCACCGT-3’
Mt-7R:5’-ARWTTTACCGGCCCTHTTA-3’
Wherein：Y represents base C or T；M represents base C or A；R represents base A or G；W represents base A or T；K represents alkali
Base T or G；H represents base A, T or C.
Fingerling checking degenerate primer amplification correctness and the taxonomic identification to unknown fingerling known to embodiment 2
1. sample collection
Sample comes from mandarin sturgeon and studies the fin ray sample collected during Upper Yangtze River creel survey in one's power, and fin ray is protected
It is stored in ethanol and takes back, if the time longer will regularly replaces ethanol.
2. sample DNA extracts
Use TIANGEN TIANamp Genomic DNA Kit (blood/cell/tissue extracting genome DNA) reagent
Box extracts species DNA to be checked.During extraction, first sample tissue (fin, muscle, fish-egg etc.) is shredded, a small amount of sample of picking is in 200 μ l
In buffer solution GA, 20 μ l Proteinase K Solutions are added, are mixed, digest 30-60min in 56 DEG C.200 μ l buffer solution GB are added, fully
It is reverse to mix, 70 DEG C of placement 10min.Add 200 μ l absolute ethyl alcohols, all liq be transferred in adsorption column, 12000rpm from
The heart 2 minutes.Waste liquid is abandoned, 500 μ l GD are added into adsorption column, 2min is centrifuged in 12000rpm.Waste liquid is abandoned, is added to adsorption column
600 μ l rinsing liquid PW, 2min is centrifuged in 12000rpm.Repeat this step.Then waste liquid is abandoned, 2min is centrifuged in 12000rpm, and
Thoroughly dried in room temperature.Adsorption column is placed in new EP pipes, 50 μ l ddH are added to adsorption column2O, centrifuged in 12000rpm
2min.DNA will be obtained and be placed in -20 DEG C of preservations.
3.PCR expands purpose fragment
Using above-mentioned 7 pairs of primers, performing PCR is entered to expand corresponding purpose fragment to the species DNA to be checked of extraction respectively.
PCR reaction systems are in every 25 μ l systems：10xbuffer 2.5 μ l, MgCl2(25mM) 1.5 μ l, dNTPs (10mM) 1 μ l are dense
Spend each 0.5 μ l of positive anti-primer for 100pmol/ μ l, the μ l of template DNA 1, Taq enzyme (5U/ μ l) 0.25 μ l, ddH2O 17.75μl。
PCR reaction conditions are 94 DEG C of pre-degeneration 3min, and 94 DEG C of denaturation 30s, 48-65 DEG C of annealing 30s, 72 DEG C of extension 2min, 30 circulate,
72 DEG C of extension 10min.Wherein, preferably 55 DEG C of annealing temperature.
4. sequencing, splicing and species identification
PCR primer is identified into clip size and gel extraction with 1% agarose gel electrophoresis, connects, converts, sequencing.Even
Junctor system is in every 5 μ l systems：Glue reclaim product 1-4 μ l, pEASY-T1 Cloning Vector 1 μ l, ddH2O 3μl；Even
Connecing reaction condition is：25 DEG C of connection 20-30min；4 DEG C preserve for converting.Wherein, linked system glue reclaim product preferably 1 μ l,
Preferably 25 DEG C connection 25min of reaction system.
5 μ l connection product is transferred in 100 μ l DH5 α competent cells, places 30min on ice, then 42 DEG C of heat shocks
40s, 2min is placed on ice.The product of conversion is added in 800 μ l LB culture mediums, 90min is cultivated in 37 DEG C of 300rpm.Take
100 μ l nutrient solutions coated plate on the LBA culture mediums of the benzyl containing ammonia, 37 DEG C are incubated overnight, and carry out choosing bacterium culture sequencing.
Obtain 7 sections of sequences are spliced, obtain complete chondriogen total order, Blast is compared on NCBI, mirror
Determine species information.As a result show：Megalobramapellegrini (thick jaw triangular bream), Acipenser sinensis (mandarin sturgeon),
Leiocassis longirostris (long-snout catfishes), Myxocyprinus asiaticus (mullet), Lepturichthys
Fimbriata (colter loach), Pelteobagrus vachelli (Pelteobagrus vachelli), the Pseudorasbora parva (wheat heads
Fish), the chondriogen qualification result of fingerling known to this 8 kinds of Coreius heterokon (copper fish) and conventional identification result phase one
Cause, it was demonstrated that the accuracy of degenerate primer qualification result；Taxonomic identification result is carried out to other four kinds fingerlings to be measured to be shown as：
Procyprisrabaudi,Erythroculter ilishaeformis,Acipenser dabryanus,Siniperca chuatsi,
The above two belong to cyprinid fish rock carp, erythroculter ilishaeformis, and both rear be followed successively by belongs to Acipenseridae fish acipenser dabryanus, Sushi sections fish mandarin fish.
According to the identification of fingerling known and to be measured, the results showed that, combined using the degenerate primer of the present invention, whole fingerlings
Amplifiable shaping band, fingerling to be measured are 4 kinds by Sequence Identification, illustrate that the degenerate primer has good versatility, are expanded
Efficiency high, and the identification of known fingerling demonstrates the accuracy of degenerate primer qualification result of the present invention.
Although above the present invention is described in detail with a general description of the specific embodiments,
Without departing from the improvement made on the basis of spirit of the invention, belong to the scope of protection of present invention.
Wherein：Sequence 1~14 represents Mt-1F, Mt-1R, Mt-2F, Mt-2R, Mt-3F, Mt-3R, Mt-4F, Mt- respectively
4R, Mt-5F, Mt-5R, Mt-6F, Mt-6R, Mt-7F, Mt-7R；Y represents base C or T；M represents base C or A；R represents base A
Or G；W represents base A or T；K represents base T or G；H represents base A, T or C.Wherein, first pair of primer extension product is 12S
RNA*, tRNA-Val, 16S RNA*, there are 2*2*3=12 kind combining forms；Second pair of primer extension product is 16S RNA*,
tRNA-Leu,ND1,tRNA-Ile,tRNA-Gln,tRNA-Met,ND2,tRNA-Trp,tRNA-Ala,tRNA-Asn,tRNA-
Cys*, there are 2*2*2=8 kind combining forms；3rd pair of primer extension product is tRNA-Asn*, tRNA-Cys, tRNA-Tyr, CO
I, tRNA-Ser, tRNA-Asp, CO II*, there are 2*2*2*2*2*2*2=128 kind combining forms；4th pair of primer extension product
For tRNA-Asp*, CO II, tRNA-Lys, ATP8, ATP6, CO III, tRNA-Gly, ND3, tRNA-Arg, ND4L, ND4*,
There are 2*2*2*2*2*2*2=128 kind combining forms；5th pair of primer extension product is ND3*, tRNA-Arg, ND4L, ND4,
TRNA-His, tRNA-Ser*, there are 2*2*2*2*2*2*2*2=256 kind combining forms；6th pair of primer extension product is tRNA-
His*, tRNA-Ser, tRNA-Leu, ND5*, there are 2*2*2*2*2*=32 kind combining forms；7th pair of primer extension product be
ND5*, ND6, tRNA-Glu, CYTB, tRNA-Thr, tRNA-Pro, D-loop, tRNA-Phe, 12SRNA*, there is 2*2*2*2*2*
2*2*3=384 kind combining forms.
1. Erythroculter ilishaeformis (erythroculter ilishaeformis) mitochondria base obtained using 7 pairs of degenerate primers
Because of fragment sequence (deepened portion is primer sequence)
Pair of primers：
TTACACATGCAAGTCTCCGCAACCCCGTGAGTATGCCCTCAATCCCCCGCCCGGGGACGAGGAGCGGGC
ATCAGGCACAAATTTTAGCCCAAGACGCCTGGCCAAGCCACACCCCCAAGGGAATTCAGCAGTGATAAACATTAAGC
CATAAGTGAAAACTTGACTCAGTCAGGGTTAAAAGGGCCGGTAAAACTCGTGCCAGCCACCGCGGTTAAACGAGAGG
CCCTAGTTGATAACACCACGGCGTAAAGGGTGGTTAAGGAGAATAACATATTTTTTAAAGCCAAATGGCCCTTTGGC
CGTCATACGCTTCTAGGTGTCCGAAGCCCAATTACACGAAAGCAGCTTTAATAAAGCCCACCTGACCCCACGAAAGC
TGAGAAACAAACTGGGATTAGATACCCCACTATGCTCAGCCATAAACCTAGACATCCAACTACAATTAGATGTCCGC
CCGGGTACTACGAGCATCAGCTTGAAACCCAAAGGACCTGACGGTGCCTCAGACCCCCCTAGAGGAGCCTGTTCTAG
AACCGATAACCCCCGTTAAACCTCACCACTTCTAGCCATCCCAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGA
AGGTAATAAAAGTAAGCAAAATGGGCACAACCCAGAACGTCAGGTCGAGGTGTAGCGCACGAAGTGGGAAGAAATGG
GCTACATTTTCTATCACAGAACACTACGAACATGCAACATGAAATAGTGCTTGAAGGAGGATTTAGTAGTAAAAAGG
AAGCAGAGTGTCCTTTTGAACCCGGCTCTGAGGCGCGTACACACCGCCCGTCACTCTCCCCTGTCAAAACGCAACAA
AGATACTTAACACTAAAGCACTGACAAGGGGAGGCAAGTCGTAACATGGTAAGTGTACCGGAAGGTGCACTTGGATT
AAACCCAGGGCGTGGCTGAGTTAGTTAAGCATCTCACTTACACCGAGAAGACATCCATGCAAGTTGGATCGCCCTGA
GCCAAACAGCTAGCTTAACCACCAATATAACCTAACAATGTTAATAACAAAACATGACCTAACACTACAAACTAAAC
CATTTTTTTACCTGAGTATGGGAGACAGAAAAGGTTCAACCCAAAGCGATAGAAAAAGTACCGCAAGGGAAAGCTGA
AAGAGAAATGAAACAACCCATATAAGCACCAAAAAACAAAGACTAAACCTTGTACCTTTTGCATCATGATTTAGCCA
GCACCCTCAGGCAAAGAGACCTTTAGTTTGAAACCCCGAAACCAGGTGAGCTACCCCGAGACAGCCTATTTAAATTT
AGGGCTAACCCGTCTCTGTGGCAAAAGAGTGGGAAGAGCTCCGGGTAGAAGTGACAGACCTACCGAACCTGGTGATA
GCTGGTTGCCTGAAAAGTGGATAGAAGTTCAGCCTCGTACACCCCAGATCAAAAAACATAACATTAAGACACAAGGG
AAACATACGAGAGTTAGTTAAAGGGGGTACAGCCCCTCTAACAAAGGATACAACCTTCACAGGAGGATAAAGATCAT
AATATATAAAACATACTGTTTTAGTGGGCCTAAAAGCAGCCATCTAAATAGAAAGCGTTAAAGCTCAGACAGAGAGA
AGTTTATTATACTGATAAAAAATCTTATTCCCCTAACAATATCAGGCTAACCCATGCCCACATGGAAGAAATTATGC
TAAAATGAGTAACAAGAAGACCTGCTCTTCTCCAAGCACAAGTGTAAGCCAGATCGGACAAACCGCTGGAAATTAAC
GAACTCAACCCAAGAGAGTAATGTGGACAACAAAAAAACCAAGAAAAACCCACAGCTAAACTAATCGTTAACCCCAC
ACTGGAGTGCTATTTTTAAAGGAAAGACTAAAAGAAAGGGAAGGAACTCGGCAAACACAAGCCTCGCCTGTTTACCA
AAAACATCGCCTCCTGCAACTAAACTGAGTATAGGAGGTCCAGCCTGCCCAGTGACTACGGGTTCAACGGCCGCGGT
ATTTTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTTAAATAGAGACCTGTATGAATGGCTAAACGAGGGCTTAA
CTGTCTCCCCCTTCAAGTCAGTGAAATTGATTTACCCGTGCAGAAGCGGGTATAACTATACAAGACGAGAAGACCCT
TTGGAGCTTAAGGTACAAAATTCAACCACGTTAAGCAACTTAATAAAAAGCAAAAACTTAGTGGAAAATGAAGTTTT
ACCTTCGGTTGGGGCGACCACGGAGGAAAAACAAGCCTCCGAGTGGAATGGGCCAAACCCCTAAAATTAAGAGAGAC
ATCTCTAAGCCACAGAACATCTGACCAAAAATGATCCGGCTAATAAAGCCGATCAACGAACCAAGTTACCCTAGGGA
TAACAGCGCAATCCTCTCCCAGAGTCCATATCGACGAGGGGGTTTACGACCTCGATGTTGGATCAGGACATCCTAAT
GGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGATCTGAGTTCAGACCGGAGCAATCCA
GGTCAGTTTCTATCT
Second pair of primer：
GTTCAACGATTAAAGTCCTACGTGATCTGAGTTCAGACCGGAGCAATCCAGGTCAGTTTCTATCTGTAA
CGCTACTTTTCCTAGTACGAAAGGATCGGAAAAGAGGGGCCCATACTTACAGCACGCCCCACCCCTAATTAATGAAA
ACAAATAAATTAAACAAAGGGAGGGCCAAAACCCCAACCGCCCGAAATAAGGACATACTGGGGTGGCAGAGCATGGT
AAATTGCGAAAGGCCTAAGCCCTTTAAACCAGAGGTTCAAATCCTCTTCCCAGTTTATGCTAAACACTCTAATAAAC
CACCTAATCAACCCCCTAGCCTATATCGTGCCTGTCCTGCTAGCAGTAGCCTTCCTAACTCTAATCGAACGTAAAGT
ATTAGGATATATACAACTACGAAAAGGACCAAATGTGGTCGGACCATATGGACTCCTACAACCTATCGCCGACGGAG
TAAAATTATTTATCAAAGAACCAGTCCGTCCCTCTACATCTTCTCCGTTCTTATTCTTAGCCACCCCCATCCTCGCA
TTAACCCTGGCCATAACTCTATGAGCACCCATACCCATACCCTACCCAGTAGTCGACCTCAACTTAGGGGTCCTCTT
TATCCTAGCCTTATCAAGCCTCGCAGTATATTCTATTCTAGGATCAGGATGGGCATCAAATTCAAAATATGCACTAA
TCGGGGCCCTACGGGCTGTAGCCCAAACAATTTCCTATGAAGTAAGCCTCGGACTAATCCTTCTATCAGTAATTATC
TTCTCAGGAGGTTATACCCTACAAACATTTAGCACAGCCCAAGAAAGTATCTGACTATTAGCCCCTGCATGACCACT
AGCCGCAATATGATATATCTCAACCCTAGCAGAAACTAACCGAGCACCATTTGACCTAACAGAGGGGGAATCAGAAC
TAGTCTCGGGCTTCAACGTAGAATATGCAGGAGGACCCTTCGCCCTTTTCTTCCTGGCCGAATACGCAAACATCCTA
TTAATAAATACCCTATCAGCTGTACTATTCCTAGGAACATCACACATCCACCACATCCCAGAACTAACAACAATCAG
CCTTATAACTAAAGCCGCACTACTTTCTATTGTATTCCTATGAGTACGAGCCTCGTACCCACGATTCCGGTATGACC
AACTAATACACCTTGTATGAAAAAACTTCCTCCCATTAACATTGGCCTTAGTACTATGACATATTGCCCTGCCAATC
GCACTAGCGGGCCTTCCCCCACAACTATAATTCAGGAACTGTGCCCGAATGCCTAGGGACCACTTTGATAGAGTGGC
CTATAGGGGCTAAAATCCCCTCAGTTCTTAGAAAGAAGGGGGTCGAACCCATGCCCAAGAGATCAAAACTCTTAGTG
CTTCCTCTACACCACTTTCTAAGATGGGGTCAGCTAATCAAGCTTTCGGGCCCATACCCCGAACATGACGGTTAAAG
TCCCTCCTCCATCAATGAACCCCTATGTACTCGCAATCCTACTATCCAGCCTAGGTTTAGGAACCACCTTAACCTTT
GCCAGCTCCCACTGACTACTGGCCTGAATAGGACTAGAAATTAACACCCTGGCAATTATCCCCCTAATAGCACAACA
CCACCACCCTCGTGCAGTAGAAGCCACCACAAAATACTTCCTAACCCAGGCCACCGCAGCCGCAATAATTATATTTG
CGAGCACAACAAACGCCTGAATCACAGGAGAATGAGACATAAACAACATATCAAACCCCCTTGCTAGCACAATGGTC
ATTACTGCCCTGGCACTTAAAATTGGACTAGCACCAATACACTTCTGAATGCCAGAAGTACTACAAGGACTAGACCT
TCTAACAGGGCTAATTTTATCAACATGACAAAAACTCGCCCCATTCGCCCTAATCATCCAAACAGCCCAAGCCGTGG
ACCCCGTACTACTAACTGCCCTAGGAATCACATCCACCTTGGTCGGAGGATGAGGAGGATTAAACCAAACCCAACTC
CGAAAAATCTTAGCCTACTCCTCAATTGCGCACATAGGCTGAATAATTATTATTCTCCAATACACCCCACAACTCAC
CCTTGTCGCCCTAGGAACATACATTTTTATGACATCCGCAGCATTCCTTACTCTAAAAACATCATCCGCCACAAAAA
TCAACACCCTCTCAATAGTCTGATCAAAAAGCCCAATCCTAACAACAACCACTGCTCTAGTACTGTTATCACTAGGT
GGCCTGCCCCCACTAACAGGATTCATGCCAAAATGACTAATCCTGCAAGAATTGGCAAAACAAAACCTTCCAGCTAC
TGCCACAATCATAGCCCTGGCTGCCTTACTAAGCCTTTACTTCTACCTGCGCCTCTGTTATGCAATAACACTCACAA
TCTCTCCCAATACAATCAACTCAATCACCCCCTGACGAACCCAAACAACCCAATCCTCCCTCCCCCTTACCCTATCT
ACCACAATCGCCCTAGGACTTTTACCTATAACCCCAGCTATTCTAATACTAGCCACCTAGGGACTTAGGATAACATC
AGACCAAGAGCCTTCAAAGCTCTAAGCAGAAGTGAAAATCTTCTAGTCCCTGATAAGACCTACAAGAGTCTATCTTG
CATTTTCTGATTGCAAATCAAATGTTTTTATTAAACTAAGGCCTTACTAGATGGGAAGGCCTCGATCCTACAAACTC
TTAGTTAACAGCTAAGCGCTCAAACCAGCGAGCATCCATCTACTTTT
3rd pair of primer：
GAAGGCCTCGATCCTACAAACTCTTAGTTAACAGCTAAGCGCTCAAACCAGCGAGCATCCATCTACTTT
TCCCGCCTATGGCCTAGTAAGGCGGGAAAAGCCCCGGCAGACTACTAATCTACGTCTCTGGATTTGCAATCCAACAT
GTTTCTTCACCACGGGGCTGATGATAAGGAGAGGACTTAAACCTCTGTCTTCGGGGCTACAACCCGCCGCCTAAGCA
CTCGGCTACCCTACCTGTGGCAATTACGCGCTGATTCTTTTCTACAAACCACAAAGACATTGGTACCCTTTATCTTG
TATTTGGTGCCTGAGCCGGAATAGTGGGAACCGCTCTAAGCCTTCTCATTCGAGCCGAACTAAGCCGACCTGGATCA
CTTCTGGGCGACGATCAAATCTATAATGTCATTGTCACTGCCCATGCCTTCGTAATAATTTTCTTTATAGTAATACC
AATTCTAATCGGAGGATTTGGAAACTGACTCGTACCACTAATAATCGGGGCACCTGATATGGCATTCCCGCGAATAA
ATAACATGAGTTTCTGACTTCTACCCCCCTCTTTTCTCCTACTGTTGGCCTCCTCTGGTGTCGAAGCCGGAGCTGGA
CAGGGTGAACAGTCTACCCACCACTCGCAGGCAACCTTGCCCACGCGGGAGCATCCGTAGACCTGACAATTTTCTCG
CTTCACCTAGCAGGTGTTTCATCAATTTTAGGTGCAATTAACTTTATCACCACAACTATTAACATAAAACCACCAGC
CATCTCTCAATATCAAACACCCCTGTTTGTTTGAGCTGTACTTGTAACAGCCGTACTTCTTCTCCTATCTCTACCAG
TCCTAGCCGCTGGAATTACTATACTCCTTACAGACCGAAATCTAAATACCACATTCTTTGACCCGGCAGGCGGGGGA
GACCCAATCCTGTATCAACACCTGTTCTGATTCTTCGGCCACCCAGAAGTTTATATTCTCATCTTACCTGGATTTGG
AATCATCTCCCACGTTGTAGCCTACTACGCAGGCAAAAAAGAACCATTCGGCTACATAGGAATGGTCTGAGCTATAA
TGGCCATTGGTCTCCTGGGATTTATTGTATGGGCTCACCATATGTTCACTGTCGGAATGGACGTAGACACTCGTGCA
TACTTCACATCCGCAACAATAATTATTGCTATCCCAACCGGTGTAAAAGTGTTTAGCTGATTAGCCACACTCCATGG
AGGGTCTATTAAATGAGAAACACCTATATTATGAGCCCTCGGATTTATTTTCCTCCTCACAGTAGGTGGACTAACAG
GAATTGTTCTAGCCAACTCATCACTCGATATTGTTCTTCACGACACATACTACGTAGTTGCACACTTCCACTACGTA
TTGTCAATAGGTGCTGTATTTGCCATCATGGCAGCTTTCGTTCACTGATTCCCCCTATTTACAAGGTACACTTTAAA
TGACACCTGAACAAAAATTCACTTCGGAGTAATATTCATTGGTGTCAATCTTACATTCTTCCCACAACACTTCCTAG
GTCTGGCAGGAATGCCACGACGATACTCTGACTACCCAGATGCCTACACCCTGTGAAATACAGTATCATCCATCGGG
TCACTTATCTCCCTAGTAGCAGTAATTATGTTCCTATTCATTTTATGAGAAGCCTTCGCCGCTAAACGGGAAGTATC
CTCAGTAGAGTTAACTATAACAAACGTAGAATGGCTTCATGGCTGTCCTCCACCATACCACACATTTGAAGAACCCG
CATTCGTCCAAGTTCAATCAAACTAACGAGAAAGGAAGGAATTGAACCCCCGTATACTGGTTTCAAGCCAGTCACAT
AACCACTCTGTCACTTTCTTCTAAAGACATTAGTAAAATACGCAAATTACATCACCTTGTCGAGGTGAAATTGCAGG
TTAAACCCCTGTATGTCTTAAGCCCTAAAGCTTAATGGCACATCCCACGCAACTAGGATTCCAAGACGCGGC
4th pair of primer：
GTTTCAAGCCAGTCACATAACCACTCTGTCACTTTCTTCTAAAGACATTAGTAAAATACGCAAATTACA
TCACCTTGTCGAGGTGAAATTGCAGGTTAAACCCCTGTATGTCTTAAGCCCTAAAGCTTAATGGCACATCCCACGCA
ACTAGGATTCCAAGACGCGGCATCACCCGTTATAGAAGAACTTCTTCACTTCCATGACCACGCCCTAATAATCGTGT
TCTTAATTAGCACTCTAGTACTTTACATTATTATTGCAATGGTTTCAACTAAGCTTACCAACAAATATATCCTAGAC
TCTCAAGAGATCGAAATCGTATGAACTATTTTACCAGCCGTAATTCTAGTTTTGATTGCTCTACCCTCCCTTCGAAT
TTTATACCTTATAGACGAAATCAATGACCCCCATCTAACAATTAAAGCCATAGGACATCAATGATATTGAAGCTATG
AGTATACAGACTACGAAGATCTGGGCTTCGACTCCTACATAATCCCGACCCAAGACCTTACACCAGGCCAATTCCGA
CTCCTAGAAACAGACCACCGGATAGTAGTCCCCATAGAATCACCAGTTCGTGTCCTAGTATCTGCCGAAGATGTATT
ACACTCCTGAGCCGTCCCATCTTTAGGCGTAAAAATAGACGCAGTGCCAGGCCGACTAAACCAAACTGCCTTCATTG
CCTCACGCCCAGGTGTGTTTTACGGACAATGCTCTGAAATTTGCGGAGCGAACCACAGCTTTATACCCATTGTAGTT
GAAGCAGTTCCACTAGAACACTTCGAGAGCTGATCCTCATTAATACTAGAAGACGCCTCACTAGAAAGCTAATTATT
GGACAAAGCGTTGGCCTTTTAAGCCAAAGTTTGGTGCCTGCCGACCACCTCTAGTGAAATGCCCCAATTAAACCCCA
ACCCCTGATTTGCAATTCTAGTATTCTCCTGAATCGTCTTTCTTACCATTATCCCAACTAAAATTTTAAATCACACC
ACACCAAATGAACCAACCCCAATAAGTGAAGAAAAACACAAAACAGAGCCCTGAGACTGACCATGATAATAAGCTTT
TTTGACCAATTCGCAAGCCCATCCTTCCTAGGCATTCCACTTATTGCTATTGCAATCGCACTACCATGACTCCTCTT
CCCAACCCCGCCATCCCGGTGAATCAACAACCGACTCATCACTCTCCAAACATGGTTCATTAACCGATTTACTAATC
AACTAATAATGCCCTTAAATGTAGGAGGACATAAATGAGCGCTTCTACTGGCCTCATTAATAGTATTCCTTATTACT
ATTAATATATTAGGCCTCCTCCCATATACTTTTACACCCACAACACAACTGTCGCTAAATATAGGATTTGCTGTACC
ACTGTGACTCGCCACCGTAATTATTGGAATGCGAAATCAGCCAACAGTTGCCCTCGGACACCTTCTACCAGAAGGAA
CGCCTATTCCTCTAATCCCCGTACTAATTATTATCGAAACAATTAGCCTATTTATTCGACCATTAGCCCTGGGTGTT
CGACTTACAGCCAACTTAACTGCCGGCCACTTATTAATTCAACTCATCGCCACAGCTGTATTTGTTTTACTGCCAAT
AATACCTACAGTAGCCATCCTAACCGCCACCGTTCTCTTCCTCCTAACACTCCTAGAAGTCGCAGTAGCAATAATTC
AAGCTTATGTATTTGTACTACTCCTAGGCCACTACCTGCAAGAAAACGTCTAATGGCCCACCAAGCACATGCATATC
ATATGGTTGATCCAAGCCCATGACCACTAACCGGAGCCGTCGGTGCTCTATTAATAACATCCGGCCTAGCAATCTGG
TTCCACTTCCACTCAACAACATTAATAACCCTTGGGGTAATTCTTTTACTTCTTACAATATTCCAATGATGACGTGA
CATTATTCGAGAAGGAACATTCCAAGGCCACCACACACCACCAGTACAAAAAGGACTACGTTATGGAATAATTCTGT
TTATTACCTCAGAGGTGTTCTTCTTCCTAGGATTCTTCTGAGCTTTCTATCACTCAAGCTTAGCACCAACACCAGAA
CTGGGAGGATGCTGACCCCCAACAGGAATCACTACATTAGACCCCTTCGAGGTTCCCCTCCTCAACACAGCCGTTCT
ACTAGCATCAGGGGTAACAGTCACATGAGCCCACCATAGCATTATAGAAGGTGAGCGAAAACAAGCCATCCAATCCC
TTGCACTCACAATTCTGCTCGGACTCTACTTCACTGCCCTTCAAGCCATAGAATACTATGAAGCACCTTTTACAATC
GCAGACGGAGTATACGGCTCTACATTCTTCGTGGCCACAGGGTTCCACGGGCTACATGTCATTATTGGATCATCATT
CCTGGCTGTTTGCCTCCTCCGCCAAATCCAATACCACTTTACATCTGAACATCATTTCGGCTTTGAAGCCGCCGCCT
GATATTGACATTTCGTTGACGTAGTATGACTATTCCTCTACGTATCCATCTATTGATGAGGCTCATAATCTTTCTAG
TATCAAAGTTAGTACAAGTGACTTCCAATCATTTAGTCTTGGTTAAACCCCAGGGAAAGATAATGAACTTAATTATA
ACTATTCTTACCATCACAATAGCCCTATCCTCAATCCTGGCAATCGTATCTTTCTGACTGCCGCAAATAAACCCAGA
CGCAGAAAAACTATCTCCCTATGAATGCGGATTCGACCCACTAGGGTCCGCCCGGCTGCCCTTTTCATTACGATTCT
TCCTAGTAGCAATCCTATTCCTCCTATTTGACCTAGAAATTGCCCTCCTCCCCCCCCTACCATGAGGAGATCAACTC
CATAACCCCACCGGAACATTCTTCTGAGCCACTACAGTCCTAATTCTGTTAACCCTAGGACTGATCTATGAGTGAAC
TCAAGGTGGCCTAGAATGAGCAGAATAGGGGGTTAGTCCAAAACAAGACCTCTGATTTCGGCTCA
5th pair of primer：
AGTACAAGTGACTTCCAATCATTTAGTCTTGGTTAAACCCCAGGGAAAGATAATGAACTTAATTATAAC
TATTCTTACCATCACAATAGCCCTATCCTCAATCCTGGCAATCGTATCTTTCTGACTGCCGCAAATAAACCCAGACG
CAGAAAAACTATCTCCCTATGAATGCGGATTCGACCCACTAGGGTCCGCCCGGCTGCCCTTTTCATTACGATTCTTC
CTAGTAGCAATCCTATTCCTCCTATTTGACCTAGAAATTGCCCTCCTCCCCCCCCTACCATGAGGAGATCAACTCCA
TAACCCCACCGGAACATTCTTCTGAGCCACTACAGTCCTAATTCTGTTAACCCTAGGACTGATCTATGAGTGAACTC
AAGGTGGCCTAGAATGAGCAGAATAGGGGGTTAGTCCAAAACAAGACCTCTGATTTCGGCTCAGAAAATTATGGTTT
AAGTCCATGATCCCCTTATGACACCAGTACACTTTAGCTTTAGCTCAGCATTCATTTTAGGACTAATAGGGCTAGCA
TTCCACCGCACCCACCTGCTCTCCGCACTCCTATGCTTAGAAGGAATAATATTATCCCTGTTTATTGCACTAGCCCT
ATGGGCCCTACAATTCGAATCGACAAGCTTCTCCGCAGCCCCTATGCTATTACTAGCCTTCTCCGCCTGCGAAGCAA
GCACGGGCCTTGCACTCCTAGTAGCCACAGCCCGAACCCATGGAACTGATCGTTTACAAAACCTTAATCTCCTACAA
TGCTAAAAGTATTAATCCCCACTGTTATATTATTCCCAACAATTTGATTAACCTCCCCCAAGTGACTATGAACAACC
ACAACTGCACATAGCCTCCTAATCGCCCTCATCAGTCTCACATGACTAAAATGGACATCAGAAACCGGATGAACCAC
ATCCAACTCATATCTAGCCACAGATCCCCTCTCAACCCCCCTCTTAGTGTTAACATGTTGACTTCTCCCATTGATAA
TTCTAGCCAGCCAAAACCACGTTAACCCAGAACCTGTCAGCCGACAACGTTTATACATCACTCTCCTCACCTCACTA
CAAACCTTTCTAATTATGGCATTCGGCGCCACCGAAATTATTATATTTTATATCATATTTGAAGCCACGCTCATCCC
AACCCTTATTATTATTACCCGATGAGGAAACCAAACCGAACGCCTCAATGCAGGGACCTATTTTCTATTCTACACCC
TAGCAGGGTCCCTCCCGCTACTAGTTGCCCTCCTTCTACTTCAACAATCTACTGGGACTCTATCCATGCTAGTTATC
CAATACTCCCAGCCATTACTTCTAGACTCCAGAGGCCACAAAATCTGATGAGCCGGCTGCTTAATTGCATTCTTAGT
GAAAATACCATTATACGGAGTACACCTCTGACTACCAAAAGCCCACGTCGAAGCCCCAGTCGCAGGATCCATGGTAC
TAGCAGCAGTTCTATTAAAACTAGGGGGACACGGAATAATACGAATAATAATTATGCTAGACCCACTATCCAAAGAA
CTAGTATACCCATTTATTATTCTGGCATTATGAGGCATTATCATGACTGGATCAATTTGCCTTCGACAAACAGACCT
CAAGTCCCTAATCGCCTACTCATCTGTCAGCCACATGGGACTTGTAGCGGGAGGAATTCTAATCCAAACCCCATGAG
GATTCTCAGGGGCTATTATTCTAATAATCGCCCACGGACTAGTGTCTTCAATACTATTCTGCTTAGCCAACACAGCC
TATGAACGAACCCACAGCCGAACCATAATTCTTGCGCGAGGACTACAAATAATTTTCCCATTAACGGCAGTATGATG
ATTCATCGCCAACCTAGCCAACCTAGCGCTACCCCCACTACCTAACCTAATAGGAGAACTAATAATTATTACAACAT
TATTCAACTGATCGCCATGAACTATTGCACTCACGGGTGCAGGAACACTAATTACAGCGGGCTACTCCTTATATATA
TTCCTTATGTCTCAACGGGGCCCAACACCAAACCATATCACTAAACTCCCACCATTTCACACCCGAGAACACCTACT
AATAGCCCTTCACCTAATCCCAGTAATTCTCCTTGTAGCAAAGCCAGAACTTATATGAGGCTGATGCTACTAGTAAG
TATAGTTTAACCAAAACATTAGATTGTGATTCTAAAAACAGGGGTTAAAATCCCCTTACTCACCAAGGAAGGACAGA
AATCAATAAGTACTGCTAATCCTTATGCACCGCGGTTAAAATCCGCGGCTTCCTCACGCTTCTGAAGGATAACAGTT
CATCCATTGGTCTTAGGAACCAAAAACTCTTGGTGCAAATCCAAGTGGAAGCTATG
6th pair of primer：
TAGGAACCAAAAACTCTTGGTGCAAATCCAAGTGGAAGCTATGAATCCAACAACCTTAATCATATCATC
CTCACTTACTCTAGTTCTTATTATCCTCATACTTCCTCTACTAACAACACTAAACCCTAAACCACAAAAACCAGAAT
GAGCAAGTACACACGTCAAAACCGCCGTCAGCACCTCGTTCTTCATCAGCCTTCTCCCACTCATAATCTTTCTAGAT
CAAGGAATAGAAAGCATCACCACAAACTGACAGTGAATAAACACACACATATTTGATACAAATATCAGCTTCAAATT
CGACCACTACTCCCTTATTTTCACCCCTATTGCCCTCTACGTCACCTGGTCTATTCTAGAGTTCGCACTATGATATA
TGCACTCTGACCCAAATATGAACCGATTTTTCAAATACCTACTTCTATTCCTAGTAGCCATAATTACCCTGGTCACA
GCCAACAACATATTCCAACTGTTCATCGGCTGAGAAGGCGTTGGAATTATATCATTCCTACTAATCGGATGGTGGTA
CGGACGGGCAGATGCTAACACAGCAGCCCTCCAGGCCGTTATCTACAACCGAGTAGGGGACATCGGACTGATTCTAA
GCATAGCATGATTCGCAATAAACCTTAACTCCTGAGAAATCCAACAAATCTTCTTCCTATCAAAAAACTTTGACATG
ACAATCCCCCTAATCGGTCTAATCCTTGCAGCAACAGGAAAATCGGCCCAATTCGGCCTACATCCCTGACTCCCTTC
TGCCATGGAGGGCCCTACGCCAGTATCCGCCCTACTCCATTCCAGCACCATGGTCGTTGCAGGAATCTTCCTATTAA
TTCGCCTCCACCCCCTCATAGAAAACAACGGCCTGGCGCTGACAATCTGCCTCTGCTTAGGAGCACTTACTACACTA
TTCACAGCCACCTGCGCCCTAACTCAAAATGATATTAAAAAAATCGTAGCTTTCTCAACATCCAGTCAACTAGGCCT
GATAATAGTTACAATTGGGTTAAACCAACCACAACTAGCATTCCTCCACATCTGCACACATGCATTCTTCAAAGCTA
TGCTATTCCTGTGCTCCGGATCAATTATTCACAGCCTAAATGATGAACAAGACATCCGAAAAATAGGAGGCCTTCAC
AACCTAATACCCGCCACCTCGACCTACCTCACAATTGGTAGCCTGGCACTAACAGGAACCCCATTCTTAGCCGGGTT
CTTCTCAAAAGATGCCATCATTGAAGCCCTAAACACCTCTTACCTTAACGCCTGAGCCCTAACCCTTACACTAATCG
CCACATCATTCACCGCGGTCTATAGCTTCCGAGTAGTATTTTTCGTAACCATGGGGTCCCCCCGATTTCTGCCACTA
TCCCCTATTAACGAAAACAATCCATTAGTCATCAACCCTATCAAACGACTTGCCTGAGGAAGCATTATTGCAGGACT
TATCATTACATCTAACTTCCTACCCTCAAAAACACCCATTATGACAATACCAACCACCCTAAAATTAGCAGCCCTCA
TAGTAACTATCATCGGACTTCTAGTGGCCATAGAACTCACAGCCATAACCAATAAACAAGTCAAAATTACCCCCACA
ATCCCCATACACCACTTTTCAAACATACTAGGGTACTTCCCCGCAATAATCCACCGACTCCCTCCCAAACTTAACTT
AACCCTAGGACAATCAGTCGCCACTAAGCTCGACCAAACATGACTTGAAATTACAGGACCAAAAGGACTAGCATTAA
CCCAAATAATGATATCAAAAATTACAAGCGACATTCAACGAGGTATAATTAAAACCTACCTAACTATTTTCCTCCTA
ACCCTGACCCTAGCCATTCTTCTAACTCTTATCTAAACGGCTCGAAGAGTCCCACGACTTAAGCCCCGAGTAAGCTC
CAACACCACAAGTAGAGTTAAAAGTAATACTCACGCACAAATAACCAACATCACCCCACCAAAAGAATATATAACAG
CCACGCCACTAACATCTCCACGTAGTATAGAAAACTCCTTCAACCCGTCAACAACTACCCAAGAACCCTCATATCAC
CCCCCTCAAAATAACCCGGCCGCTAACACAACACCTAGAAGGTAGACTAACACATACCCAGCTACAGAACGACTTCC
CCAGGCCTCCGGAAAAGGCTCAGCAGCCAAGGCTGCTGAATAGGCAAACACCACGAGCATCCCCCCCAAATAAATTA
AAAAGAGAACCAAAGACAAGAAAGAACCCCCATAACCAACTAAAATCCCACACCCAACCCCCGCTGCTACTACCAAG
CCTAAAGCAGCAAAATAAGGCGTAGGATTAGAAGCAACAGCAATTAAACCCACAACCAGAGCTACCAATAACAAAAA
CATAAAATAAGTCATAATTCTTGCTCGGATTCTAACCGAGACCAGTGACTTGAAGAACCACCGTTGT
7th pair of primer：
TGACTTGAAGAACCACCGTTGTAGTTCAACTACAAGAACAATAATGGCAAGCCTACGAAAAACCCACCC
ACTAATAAAAATCGCCAACGACGCACTAGTTGACCTCCCCACACCATCTAACATTTCCGTATGATGAAATTTCGGGT
CTCTCCTGGGGTTGTGTTTAATTACTCAAATCCTAACCGGATTGTTCCTAGCCATACACTATACCTCTGACATCTCA
ACCGCATTTTCATCAGTAGTCCACATCTGCCGAGACGTGAACTATGGCTGACTTATCCGTAATATTCACGCCAACGG
GGCATCATTCTTTTTTATCTGTATTTATATACACATTGCCCGCGGCCTATACTATGGATCCTACCTGTATAAAGAAA
CCTGAAACATTGGGGTAGTCCTTCTCCTGTTAGTTATAATAACAGCCTTTGTTGGCTACGTCCTTCCATGAGGACAA
ATATCCTTTTGAGGTGCCACAGTAATTACAAATCTACTATCAGCAGTCCCCTACATAGGGGACACCCTCGTTCAATG
AATCTGAGGTGGCTTCTCAGTAGACAACGCGACACTAACACGGATCTTCGCATTCCACTTCCTATTACCATTCATCG
TCGCCGCCGCGACCATCCTACACCTACTTTTCCTCCACGAAACAGGCTCGAACAACCCAGCCGGGCTAAACTCCGAC
GCAGACAAAATCTCCTTCCACCCATACTTCTCATATAAAGACCTTCTAGGATTTGTAGTGATATTATTAGCCCTCAC
ATCATTAGCACTATTCTCTCCAAATCTCCTGGGAGACCCAGAGAATTTCACCCCTGCAAACCCACTAGTCACCCCTC
CACATATCAAGCCAGAATGATACTTCCTATTTGCTTACGCCATTCTACGGTCTATCCCGAACAAACTAGGAGGGGTC
CTTGCATTGCTATTCTCTATTTTAGTCCTAATGGTGGTACCAATTTTACACACCTCAAAACAACGAGGACTAACATT
CCGCCCGATCACTCAATTCCTATTCTGAACCCTAGTAGCAGATATAATTATCCTAACATGAATTGGAGGCATACCTG
TAGAACATCCATACATTATCATCGGACAAATTGCATCAGTCCTTTACTTCGCATTATTCCTCATCCTCACTCCACTA
GCAGGATGACTAGAAAATAAAGCACTAAAATGAGCTTGCCCTAGTAGCTTAGTATTAAAGCATCGGTCTTGTAATCC
GAAGATCGGAGGTTATAGTAACCTATATGGTTTAATACATATATATGTATTATATTACATAATGCATTAGTACAGAT
ATATGTATTATCACCATTCATTTATATTAACCATAAAGCAAGTACTAACGTTCAAGACGTACATAAACCAAAATATT
AAAACTCACAAATAATTTATTTAGACCCGGGAAATAGATTATTCCCCTATAATTGGCCCTCAAATATTTCCTTGAAA
TAATCAACTAGGATTTAACTCAACCATATTAATGTAGTAAGAGACCACCAACTGGTTTATATTAAGGTATATCCTGC
ATGATAGGATCAGGGACACAAATTGTGGGGGTAACACAGAATGAACTATTACTTGCATCTGGCTTCAATCTCAGGAA
CATAACTGTAAAATTCCACCCTCGGATAATTATGTCTGGCATATGGTTAAATGATGTGAGTACATACTCCTCATTAA
CCCCACATGCCGGGCATTCTTTTATATGCATAGGGGTTCTCCTTTTTGGTTACCTTTCACCTTGCATCTCAGAGTGC
AGGCTCAAATGACAAATCAAGGTTGAACATATTCCTTGCTTGAATTCGAGTAGGTCAATTATTAAAAGACATAACTT
AAGAATTACATACTTTTAACTCAAGTGCATAACACATTCATTCCTTCTTCAACTTACCCCTATATATATGCCCCCCC
TCTCGGTTTCTGCGCGACAAACCCCCCTACCCCCTACGCTCAGCAAATCCTGTTATCCTTGTCAAACCCCGAAACCA
AGGAAGGTTCGAGAACGTATAAGCTAGCAAGTTGATTTATGGGCTAGCTATCCGCATTATATATATATATATACATA
CACATCGCATTAATTTGCCGCAAATTTCTCCAAATATTGGCCTAAAAGTCCCTATTAAATTTTTAGGGCGCGCCCCA
ATGCTAAAAAGTCCAACATTAAAAAGCGCTAGCGTAGCTTAATACAAAGCATAACACTGAAGATGTTAAGATGGGCC
CTGAGAAGCTCCGCATGCATAAAGGCATGGTCCCGACCTTATTATCAGCTCTTACCCGACTTACACATGCAAGTCTC
CGCAGTCCCGTGAGTATGCCCTCAATCCCCCGCCCGGGGACGAGGAGCGGGCATCAGGCACAAATTTTAGCCCAAGA
CGCCTGGCCAAGCCACACCCCCAAGGGAATTCAGCAGTGATAAACATTAAGCCATAAGTGAAAACTTGACTCAGTCA
GGGCTAAGAGGGCCGGTAAAACT
Qiao Zui Hong Erythroculter mitochondrial DNA total orders：
CGTGCCAGCCACCGCGGTTAAACGAGAGGCCCTAGTTGATAACACCACGGCGTAAAGGGTGGTTAAGGA
GAATAACATATTTTTTAAAGCCAAATGGCCCTTTGGCCGTCATACGCTTCTAGGTGTCCGAAGCCCAATTACACGAA
AGCAGCTTTAATAAAGCCCACCTGACCCCACGAAAGCTGAGAAACAAACTGGGATTAGATACCCCACTATGCTCAGC
CATAAACCTAGACATCCAACTACAATTAGATGTCCGCCCGGGTACTACGAGCATCAGCTTGAAACCCAAAGGACCTG
ACGGTGCCTCAGACCCCCCTAGAGGAGCCTGTTCTAGAACCGATAACCCCCGTTAAACCTCACCACTTCTAGCCATC
CCAGCCTATATACCGCCGTCGTCAGCTTACCCTGTGAAGGTAATAAAAGTAAGCAAAATGGGCACAACCCAGAACGT
CAGGTCGAGGTGTAGCGCACGAAGTGGGAAGAAATGGGCTACATTTTCTATCACAGAACACTACGAACATGCAACAT
GAAATAGTGCTTGAAGGAGGATTTAGTAGTAAAAAGGAAGCAGAGTGTCCTTTTGAACCCGGCTCTGAGGCGCGTAC
ACACCGCCCGTCACTCTCCCCTGTCAAAACGCAACAAAGATACTTAACACTAAAGCACTGACAAGGGGAGGCAAGTC
GTAACATGGTAAGTGTACCGGAAGGTGCACTTGGATTAAACCCAGGGCGTGGCTGAGTTAGTTAAGCATCTCACTTA
CACCGAGAAGACATCCATGCAAGTTGGATCGCCCTGAGCCAAACAGCTAGCTTAACCACCAATATAACCTAACAATG
TTAATAACAAAACATGACCTAACACTACAAACTAAACCATTTTTTTACCTGAGTATGGGAGACAGAAAAGGTTCAAC
CCAAAGCGATAGAAAAAGTACCGCAAGGGAAAGCTGAAAGAGAAATGAAACAACCCATATAAGCACCAAAAAACAAA
GACTAAACCTTGTACCTTTTGCATCATGATTTAGCCAGCACCCTCAGGCAAAGAGACCTTTAGTTTGAAACCCCGAA
ACCAGGTGAGCTACCCCGAGACAGCCTATTTAAATTTAGGGCTAACCCGTCTCTGTGGCAAAAGAGTGGGAAGAGCT
CCGGGTAGAAGTGACAGACCTACCGAACCTGGTGATAGCTGGTTGCCTGAAAAGTGGATAGAAGTTCAGCCTCGTAC
ACCCCAGATCAAAAAACATAACATTAAGACACAAGGGAAACATACGAGAGTTAGTTAAAGGGGGTACAGCCCCTCTA
ACAAAGGATACAACCTTCACAGGAGGATAAAGATCATAATATATAAAACATACTGTTTTAGTGGGCCTAAAAGCAGC
CATCTAAATAGAAAGCGTTAAAGCTCAGACAGAGAGAAGTTTATTATACTGATAAAAAATCTTATTCCCCTAACAAT
ATCAGGCTAACCCATGCCCACATGGAAGAAATTATGCTAAAATGAGTAACAAGAAGACCTGCTCTTCTCCAAGCACA
AGTGTAAGCCAGATCGGACAAACCGCTGGAAATTAACGAACTCAACCCAAGAGAGTAATGTGGACAACAAAAAAACC
AAGAAAAACCCACAGCTAAACTAATCGTTAACCCCACACTGGAGTGCTATTTTTAAAGGAAAGACTAAAAGAAAGGG
AAGGAACTCGGCAAACACAAGCCTCGCCTGTTTACCAAAAACATCGCCTCCTGCAACTAAACTGAGTATAGGAGGTC
CAGCCTGCCCAGTGACTACGGGTTCAACGGCCGCGGTATTTTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTTA
AATAGAGACCTGTATGAATGGCTAAACGAGGGCTTAACTGTCTCCCCCTTCAAGTCAGTGAAATTGATTTACCCGTG
CAGAAGCGGGTATAACTATACAAGACGAGAAGACCCTTTGGAGCTTAAGGTACAAAATTCAACCACGTTAAGCAACT
TAATAAAAAGCAAAAACTTAGTGGAAAATGAAGTTTTACCTTCGGTTGGGGCGACCACGGAGGAAAAACAAGCCTCC
GAGTGGAATGGGCCAAACCCCTAAAATTAAGAGAGACATCTCTAAGCCACAGAACATCTGACCAAAAATGATCCGGC
TAATAAAGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCAATCCTCTCCCAGAGTCCATATCGACGAGGG
GGTTTACGACCTCGATGTTGGATCAGGACATCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAA
AGTCCTACGTGATCTGAGTTCAGACCGGAGCAATCCAGGTCAGTTTCTATCTGTAACGCTACTTTTCCTAGTACGAA
AGGATCGGAAAAGAGGGGCCCATACTTACAGCACGCCCCACCCCTAATTAATGAAAACAAATAAATTAAACAAAGGG
AGGGCCAAAACCCCAACCGCCCGAAATAAGGACATACTGGGGTGGCAGAGCATGGTAAATTGCGAAAGGCCTAAGCC
CTTTAAACCAGAGGTTCAAATCCTCTTCCCAGTTTATGCTAAACACTCTAATAAACCACCTAATCAACCCCCTAGCC
TATATCGTGCCTGTCCTGCTAGCAGTAGCCTTCCTAACTCTAATCGAACGTAAAGTATTAGGATATATACAACTACG
AAAAGGACCAAATGTGGTCGGACCATATGGACTCCTACAACCTATCGCCGACGGAGTAAAATTATTTATCAAAGAAC
CAGTCCGTCCCTCTACATCTTCTCCGTTCTTATTCTTAGCCACCCCCATCCTCGCATTAACCCTGGCCATAACTCTA
TGAGCACCCATACCCATACCCTACCCAGTAGTCGACCTCAACTTAGGGGTCCTCTTTATCCTAGCCTTATCAAGCCT
CGCAGTATATTCTATTCTAGGATCAGGATGGGCATCAAATTCAAAATATGCACTAATCGGGGCCCTACGGGCTGTAG
CCCAAACAATTTCCTATGAAGTAAGCCTCGGACTAATCCTTCTATCAGTAATTATCTTCTCAGGAGGTTATACCCTA
CAAACATTTAGCACAGCCCAAGAAAGTATCTGACTATTAGCCCCTGCATGACCACTAGCCGCAATATGATATATCTC
AACCCTAGCAGAAACTAACCGAGCACCATTTGACCTAACAGAGGGGGAATCAGAACTAGTCTCGGGCTTCAACGTAG
AATATGCAGGAGGACCCTTCGCCCTTTTCTTCCTGGCCGAATACGCAAACATCCTATTAATAAATACCCTATCAGCT
GTACTATTCCTAGGAACATCACACATCCACCACATCCCAGAACTAACAACAATCAGCCTTATAACTAAAGCCGCACT
ACTTTCTATTGTATTCCTATGAGTACGAGCCTCGTACCCACGATTCCGGTATGACCAACTAATACACCTTGTATGAA
AAAACTTCCTCCCATTAACATTGGCCTTAGTACTATGACATATTGCCCTGCCAATCGCACTAGCGGGCCTTCCCCCA
CAACTATAATTCAGGAACTGTGCCCGAATGCCTAGGGACCACTTTGATAGAGTGGCCTATAGGGGCTAAAATCCCCT
CAGTTCTTAGAAAGAAGGGGGTCGAACCCATGCCCAAGAGATCAAAACTCTTAGTGCTTCCTCTACACCACTTTCTA
AGATGGGGTCAGCTAATCAAGCTTTCGGGCCCATACCCCGAACATGACGGTTAAAGTCCCTCCTCCATCAATGAACC
CCTATGTACTCGCAATCCTACTATCCAGCCTAGGTTTAGGAACCACCTTAACCTTTGCCAGCTCCCACTGACTACTG
GCCTGAATAGGACTAGAAATTAACACCCTGGCAATTATCCCCCTAATAGCACAACACCACCACCCTCGTGCAGTAGA
AGCCACCACAAAATACTTCCTAACCCAGGCCACCGCAGCCGCAATAATTATATTTGCGAGCACAACAAACGCCTGAA
TCACAGGAGAATGAGACATAAACAACATATCAAACCCCCTTGCTAGCACAATGGTCATTACTGCCCTGGCACTTAAA
ATTGGACTAGCACCAATACACTTCTGAATGCCAGAAGTACTACAAGGACTAGACCTTCTAACAGGGCTAATTTTATC
AACATGACAAAAACTCGCCCCATTCGCCCTAATCATCCAAACAGCCCAAGCCGTGGACCCCGTACTACTAACTGCCC
TAGGAATCACATCCACCTTGGTCGGAGGATGAGGAGGATTAAACCAAACCCAACTCCGAAAAATCTTAGCCTACTCC
TCAATTGCGCACATAGGCTGAATAATTATTATTCTCCAATACACCCCACAACTCACCCTTGTCGCCCTAGGAACATA
CATTTTTATGACATCCGCAGCATTCCTTACTCTAAAAACATCATCCGCCACAAAAATCAACACCCTCTCAATAGTCT
GATCAAAAAGCCCAATCCTAACAACAACCACTGCTCTAGTACTGTTATCACTAGGTGGCCTGCCCCCACTAACAGGA
TTCATGCCAAAATGACTAATCCTGCAAGAATTGGCAAAACAAAACCTTCCAGCTACTGCCACAATCATAGCCCTGGC
TGCCTTACTAAGCCTTTACTTCTACCTGCGCCTCTGTTATGCAATAACACTCACAATCTCTCCCAATACAATCAACT
CAATCACCCCCTGACGAACCCAAACAACCCAATCCTCCCTCCCCCTTACCCTATCTACCACAATCGCCCTAGGACTT
TTACCTATAACCCCAGCTATTCTAATACTAGCCACCTAGGGACTTAGGATAACATCAGACCAAGAGCCTTCAAAGCT
CTAAGCAGAAGTGAAAATCTTCTAGTCCCTGATAAGACCTACAAGAGTCTATCTTGCATTTTCTGATTGCAAATCAA
ATGTTTTTATTAAACTAAGGCCTTACTAGATGGGAAGGCCTCGATCCTACAAACTCTTAGTTAACAGCTAAGCGCTC
AAACCAGCGAGCATCCATCTACTTTTCCCGCCTATGGCCTAGTAAGGCGGGAAAAGCCCCGGCAGACTACTAATCTA
CGTCTCTGGATTTGCAATCCAACATGTTTCTTCACCACGGGGCTGATGATAAGGAGAGGACTTAAACCTCTGTCTTC
GGGGCTACAACCCGCCGCCTAAGCACTCGGCTACCCTACCTGTGGCAATTACGCGCTGATTCTTTTCTACAAACCAC
AAAGACATTGGTACCCTTTATCTTGTATTTGGTGCCTGAGCCGGAATAGTGGGAACCGCTCTAAGCCTTCTCATTCG
AGCCGAACTAAGCCGACCTGGATCACTTCTGGGCGACGATCAAATCTATAATGTCATTGTCACTGCCCATGCCTTCG
TAATAATTTTCTTTATAGTAATACCAATTCTAATCGGAGGATTTGGAAACTGACTCGTACCACTAATAATCGGGGCA
CCTGATATGGCATTCCCGCGAATAAATAACATGAGTTTCTGACTTCTACCCCCCTCTTTTCTCCTACTGTTGGCCTC
CTCTGGTGTCGAAGCCGGAGCTGGACAGGGTGAACAGTCTACCCACCACTCGCAGGCAACCTTGCCCACGCGGGAGC
ATCCGTAGACCTGACAATTTTCTCGCTTCACCTAGCAGGTGTTTCATCAATTTTAGGTGCAATTAACTTTATCACCA
CAACTATTAACATAAAACCACCAGCCATCTCTCAATATCAAACACCCCTGTTTGTTTGAGCTGTACTTGTAACAGCC
GTACTTCTTCTCCTATCTCTACCAGTCCTAGCCGCTGGAATTACTATACTCCTTACAGACCGAAATCTAAATACCAC
ATTCTTTGACCCGGCAGGCGGGGGAGACCCAATCCTGTATCAACACCTGTTCTGATTCTTCGGCCACCCAGAAGTTT
ATATTCTCATCTTACCTGGATTTGGAATCATCTCCCACGTTGTAGCCTACTACGCAGGCAAAAAAGAACCATTCGGC
TACATAGGAATGGTCTGAGCTATAATGGCCATTGGTCTCCTGGGATTTATTGTATGGGCTCACCATATGTTCACTGT
CGGAATGGACGTAGACACTCGTGCATACTTCACATCCGCAACAATAATTATTGCTATCCCAACCGGTGTAAAAGTGT
TTAGCTGATTAGCCACACTCCATGGAGGGTCTATTAAATGAGAAACACCTATATTATGAGCCCTCGGATTTATTTTC
CTCCTCACAGTAGGTGGACTAACAGGAATTGTTCTAGCCAACTCATCACTCGATATTGTTCTTCACGACACATACTA
CGTAGTTGCACACTTCCACTACGTATTGTCAATAGGTGCTGTATTTGCCATCATGGCAGCTTTCGTTCACTGATTCC
CCCTATTTACAAGGTACACTTTAAATGACACCTGAACAAAAATTCACTTCGGAGTAATATTCATTGGTGTCAATCTT
ACATTCTTCCCACAACACTTCCTAGGTCTGGCAGGAATGCCACGACGATACTCTGACTACCCAGATGCCTACACCCT
GTGAAATACAGTATCATCCATCGGGTCACTTATCTCCCTAGTAGCAGTAATTATGTTCCTATTCATTTTATGAGAAG
CCTTCGCCGCTAAACGGGAAGTATCCTCAGTAGAGTTAACTATAACAAACGTAGAATGGCTTCATGGCTGTCCTCCA
CCATACCACACATTTGAAGAACCCGCATTCGTCCAAGTTCAATCAAACTAACGAGAAAGGAAGGAATTGAACCCCCG
TATACTGGTTTCAAGCCAGTCACATAACCACTCTGTCACTTTCTTCTAAAGACATTAGTAAAATACGCAAATTACAT
CACCTTGTCGAGGTGAAATTGCAGGTTAAACCCCTGTATGTCTTAAGCCCTAAAGCTTAATGGCACATCCCACGCAA
CTAGGATTCCAAGACGCGGCATCACCCGTTATAGAAGAACTTCTTCACTTCCATGACCACGCCCTAATAATCGTGTT
CTTAATTAGCACTCTAGTACTTTACATTATTATTGCAATGGTTTCAACTAAGCTTACCAACAAATATATCCTAGACT
CTCAAGAGATCGAAATCGTATGAACTATTTTACCAGCCGTAATTCTAGTTTTGATTGCTCTACCCTCCCTTCGAATT
TTATACCTTATAGACGAAATCAATGACCCCCATCTAACAATTAAAGCCATAGGACATCAATGATATTGAAGCTATGA
GTATACAGACTACGAAGATCTGGGCTTCGACTCCTACATAATCCCGACCCAAGACCTTACACCAGGCCAATTCCGAC
TCCTAGAAACAGACCACCGGATAGTAGTCCCCATAGAATCACCAGTTCGTGTCCTAGTATCTGCCGAAGATGTATTA
CACTCCTGAGCCGTCCCATCTTTAGGCGTAAAAATAGACGCAGTGCCAGGCCGACTAAACCAAACTGCCTTCATTGC
CTCACGCCCAGGTGTGTTTTACGGACAATGCTCTGAAATTTGCGGAGCGAACCACAGCTTTATACCCATTGTAGTTG
AAGCAGTTCCACTAGAACACTTCGAGAGCTGATCCTCATTAATACTAGAAGACGCCTCACTAGAAAGCTAATTATTG
GACAAAGCGTTGGCCTTTTAAGCCAAAGTTTGGTGCCTGCCGACCACCTCTAGTGAAATGCCCCAATTAAACCCCAA
CCCCTGATTTGCAATTCTAGTATTCTCCTGAATCGTCTTTCTTACCATTATCCCAACTAAAATTTTAAATCACACCA
CACCAAATGAACCAACCCCAATAAGTGAAGAAAAACACAAAACAGAGCCCTGAGACTGACCATGATAATAAGCTTTT
TTGACCAATTCGCAAGCCCATCCTTCCTAGGCATTCCACTTATTGCTATTGCAATCGCACTACCATGACTCCTCTTC
CCAACCCCGCCATCCCGGTGAATCAACAACCGACTCATCACTCTCCAAACATGGTTCATTAACCGATTTACTAATCA
ACTAATAATGCCCTTAAATGTAGGAGGACATAAATGAGCGCTTCTACTGGCCTCATTAATAGTATTCCTTATTACTA
TTAATATATTAGGCCTCCTCCCATATACTTTTACACCCACAACACAACTGTCGCTAAATATAGGATTTGCTGTACCA
CTGTGACTCGCCACCGTAATTATTGGAATGCGAAATCAGCCAACAGTTGCCCTCGGACACCTTCTACCAGAAGGAAC
GCCTATTCCTCTAATCCCCGTACTAATTATTATCGAAACAATTAGCCTATTTATTCGACCATTAGCCCTGGGTGTTC
GACTTACAGCCAACTTAACTGCCGGCCACTTATTAATTCAACTCATCGCCACAGCTGTATTTGTTTTACTGCCAATA
ATACCTACAGTAGCCATCCTAACCGCCACCGTTCTCTTCCTCCTAACACTCCTAGAAGTCGCAGTAGCAATAATTCA
AGCTTATGTATTTGTACTACTCCTAGGCCACTACCTGCAAGAAAACGTCTAATGGCCCACCAAGCACATGCATATCA
TATGGTTGATCCAAGCCCATGACCACTAACCGGAGCCGTCGGTGCTCTATTAATAACATCCGGCCTAGCAATCTGGT
TCCACTTCCACTCAACAACATTAATAACCCTTGGGGTAATTCTTTTACTTCTTACAATATTCCAATGATGACGTGAC
ATTATTCGAGAAGGAACATTCCAAGGCCACCACACACCACCAGTACAAAAAGGACTACGTTATGGAATAATTCTGTT
TATTACCTCAGAGGTGTTCTTCTTCCTAGGATTCTTCTGAGCTTTCTATCACTCAAGCTTAGCACCAACACCAGAAC
TGGGAGGATGCTGACCCCCAACAGGAATCACTACATTAGACCCCTTCGAGGTTCCCCTCCTCAACACAGCCGTTCTA
CTAGCATCAGGGGTAACAGTCACATGAGCCCACCATAGCATTATAGAAGGTGAGCGAAAACAAGCCATCCAATCCCT
TGCACTCACAATTCTGCTCGGACTCTACTTCACTGCCCTTCAAGCCATAGAATACTATGAAGCACCTTTTACAATCG
CAGACGGAGTATACGGCTCTACATTCTTCGTGGCCACAGGGTTCCACGGGCTACATGTCATTATTGGATCATCATTC
CTGGCTGTTTGCCTCCTCCGCCAAATCCAATACCACTTTACATCTGAACATCATTTCGGCTTTGAAGCCGCCGCCTG
ATATTGACATTTCGTTGACGTAGTATGACTATTCCTCTACGTATCCATCTATTGATGAGGCTCATAATCTTTCTAGT
ATCAAAGTTAGTACAAGTGACTTCCAATCATTTAGTCTTGGTTAAACCCCAGGGAAAGATAATGAACTTAATTATAA
CTATTCTTACCATCACAATAGCCCTATCCTCAATCCTGGCAATCGTATCTTTCTGACTGCCGCAAATAAACCCAGAC
GCAGAAAAACTATCTCCCTATGAATGCGGATTCGACCCACTAGGGTCCGCCCGGCTGCCCTTTTCATTACGATTCTT
CCTAGTAGCAATCCTATTCCTCCTATTTGACCTAGAAATTGCCCTCCTCCCCCCCCTACCATGAGGAGATCAACTCC
ATAACCCCACCGGAACATTCTTCTGAGCCACTACAGTCCTAATTCTGTTAACCCTAGGACTGATCTATGAGTGAACT
CAAGGTGGCCTAGAATGAGCAGAATAGGGGGTTAGTCCAAAACAAGACCTCTGATTTCGGCTCAGAAAATTATGGTT
TAAGTCCATGATCCCCTTATGACACCAGTACACTTTAGCTTTAGCTCAGCATTCATTTTAGGACTAATAGGGCTAGC
ATTCCACCGCACCCACCTGCTCTCCGCACTCCTATGCTTAGAAGGAATAATATTATCCCTGTTTATTGCACTAGCCC
TATGGGCCCTACAATTCGAATCGACAAGCTTCTCCGCAGCCCCTATGCTATTACTAGCCTTCTCCGCCTGCGAAGCA
AGCACGGGCCTTGCACTCCTAGTAGCCACAGCCCGAACCCATGGAACTGATCGTTTACAAAACCTTAATCTCCTACA
ATGCTAAAAGTATTAATCCCCACTGTTATATTATTCCCAACAATTTGATTAACCTCCCCCAAGTGACTATGAACAAC
CACAACTGCACATAGCCTCCTAATCGCCCTCATCAGTCTCACATGACTAAAATGGACATCAGAAACCGGATGAACCA
CATCCAACTCATATCTAGCCACAGATCCCCTCTCAACCCCCCTCTTAGTGTTAACATGTTGACTTCTCCCATTGATA
ATTCTAGCCAGCCAAAACCACGTTAACCCAGAACCTGTCAGCCGACAACGTTTATACATCACTCTCCTCACCTCACT
ACAAACCTTTCTAATTATGGCATTCGGCGCCACCGAAATTATTATATTTTATATCATATTTGAAGCCACGCTCATCC
CAACCCTTATTATTATTACCCGATGAGGAAACCAAACCGAACGCCTCAATGCAGGGACCTATTTTCTATTCTACACC
CTAGCAGGGTCCCTCCCGCTACTAGTTGCCCTCCTTCTACTTCAACAATCTACTGGGACTCTATCCATGCTAGTTAT
CCAATACTCCCAGCCATTACTTCTAGACTCCAGAGGCCACAAAATCTGATGAGCCGGCTGCTTAATTGCATTCTTAG
TGAAAATACCATTATACGGAGTACACCTCTGACTACCAAAAGCCCACGTCGAAGCCCCAGTCGCAGGATCCATGGTA
CTAGCAGCAGTTCTATTAAAACTAGGGGGACACGGAATAATACGAATAATAATTATGCTAGACCCACTATCCAAAGA
ACTAGTATACCCATTTATTATTCTGGCATTATGAGGCATTATCATGACTGGATCAATTTGCCTTCGACAAACAGACC
TCAAGTCCCTAATCGCCTACTCATCTGTCAGCCACATGGGACTTGTAGCGGGAGGAATTCTAATCCAAACCCCATGA
GGATTCTCAGGGGCTATTATTCTAATAATCGCCCACGGACTAGTGTCTTCAATACTATTCTGCTTAGCCAACACAGC
CTATGAACGAACCCACAGCCGAACCATAATTCTTGCGCGAGGACTACAAATAATTTTCCCATTAACGGCAGTATGAT
GATTCATCGCCAACCTAGCCAACCTAGCGCTACCCCCACTACCTAACCTAATAGGAGAACTAATAATTATTACAACA
TTATTCAACTGATCGCCATGAACTATTGCACTCACGGGTGCAGGAACACTAATTACAGCGGGCTACTCCTTATATAT
ATTCCTTATGTCTCAACGGGGCCCAACACCAAACCATATCACTAAACTCCCACCATTTCACACCCGAGAACACCTAC
TAATAGCCCTTCACCTAATCCCAGTAATTCTCCTTGTAGCAAAGCCAGAACTTATATGAGGCTGATGCTACTAGTAA
GTATAGTTTAACCAAAACATTAGATTGTGATTCTAAAAACAGGGGTTAAAATCCCCTTACTCACCAAGGAAGGACAG
AAATCAATAAGTACTGCTAATCCTTATGCACCGCGGTTAAAATCCGCGGCTTCCTCACGCTTCTGAAGGATAACAGT
TCATCCATTGGTCTTAGGAACCAAAAACTCTTGGTGCAAATCCAAGTGGAAGCTATGAATCCAACAACCTTAATCAT
ATCATCCTCACTTACTCTAGTTCTTATTATCCTCATACTTCCTCTACTAACAACACTAAACCCTAAACCACAAAAAC
CAGAATGAGCAAGTACACACGTCAAAACCGCCGTCAGCACCTCGTTCTTCATCAGCCTTCTCCCACTCATAATCTTT
CTAGATCAAGGAATAGAAAGCATCACCACAAACTGACAGTGAATAAACACACACATATTTGATACAAATATCAGCTT
CAAATTCGACCACTACTCCCTTATTTTCACCCCTATTGCCCTCTACGTCACCTGGTCTATTCTAGAGTTCGCACTAT
GATATATGCACTCTGACCCAAATATGAACCGATTTTTCAAATACCTACTTCTATTCCTAGTAGCCATAATTACCCTG
GTCACAGCCAACAACATATTCCAACTGTTCATCGGCTGAGAAGGCGTTGGAATTATATCATTCCTACTAATCGGATG
GTGGTACGGACGGGCAGATGCTAACACAGCAGCCCTCCAGGCCGTTATCTACAACCGAGTAGGGGACATCGGACTGA
TTCTAAGCATAGCATGATTCGCAATAAACCTTAACTCCTGAGAAATCCAACAAATCTTCTTCCTATCAAAAAACTTT
GACATGACAATCCCCCTAATCGGTCTAATCCTTGCAGCAACAGGAAAATCGGCCCAATTCGGCCTACATCCCTGACT
CCCTTCTGCCATGGAGGGCCCTACGCCAGTATCCGCCCTACTCCATTCCAGCACCATGGTCGTTGCAGGAATCTTCC
TATTAATTCGCCTCCACCCCCTCATAGAAAACAACGGCCTGGCGCTGACAATCTGCCTCTGCTTAGGAGCACTTACT
ACACTATTCACAGCCACCTGCGCCCTAACTCAAAATGATATTAAAAAAATCGTAGCTTTCTCAACATCCAGTCAACT
AGGCCTGATAATAGTTACAATTGGGTTAAACCAACCACAACTAGCATTCCTCCACATCTGCACACATGCATTCTTCA
AAGCTATGCTATTCCTGTGCTCCGGATCAATTATTCACAGCCTAAATGATGAACAAGACATCCGAAAAATAGGAGGC
CTTCACAACCTAATACCCGCCACCTCGACCTACCTCACAATTGGTAGCCTGGCACTAACAGGAACCCCATTCTTAGC
CGGGTTCTTCTCAAAAGATGCCATCATTGAAGCCCTAAACACCTCTTACCTTAACGCCTGAGCCCTAACCCTTACAC
TAATCGCCACATCATTCACCGCGGTCTATAGCTTCCGAGTAGTATTTTTCGTAACCATGGGGTCCCCCCGATTTCTG
CCACTATCCCCTATTAACGAAAACAATCCATTAGTCATCAACCCTATCAAACGACTTGCCTGAGGAAGCATTATTGC
AGGACTTATCATTACATCTAACTTCCTACCCTCAAAAACACCCATTATGACAATACCAACCACCCTAAAATTAGCAG
CCCTCATAGTAACTATCATCGGACTTCTAGTGGCCATAGAACTCACAGCCATAACCAATAAACAAGTCAAAATTACC
CCCACAATCCCCATACACCACTTTTCAAACATACTAGGGTACTTCCCCGCAATAATCCACCGACTCCCTCCCAAACT
TAACTTAACCCTAGGACAATCAGTCGCCACTAAGCTCGACCAAACATGACTTGAAATTACAGGACCAAAAGGACTAG
CATTAACCCAAATAATGATATCAAAAATTACAAGCGACATTCAACGAGGTATAATTAAAACCTACCTAACTATTTTC
CTCCTAACCCTGACCCTAGCCATTCTTCTAACTCTTATCTAAACGGCTCGAAGAGTCCCACGACTTAAGCCCCGAGT
AAGCTCCAACACCACAAGTAGAGTTAAAAGTAATACTCACGCACAAATAACCAACATCACCCCACCAAAAGAATATA
TAACAGCCACGCCACTAACATCTCCACGTAGTATAGAAAACTCCTTCAACCCGTCAACAACTACCCAAGAACCCTCA
TATCACCCCCCTCAAAATAACCCGGCCGCTAACACAACACCTAGAAGGTAGACTAACACATACCCAGCTACAGAACG
ACTTCCCCAGGCCTCCGGAAAAGGCTCAGCAGCCAAGGCTGCTGAATAGGCAAACACCACGAGCATCCCCCCCAAAT
AAATTAAAAAGAGAACCAAAGACAAGAAAGAACCCCCATAACCAACTAAAATCCCACACCCAACCCCCGCTGCTACT
ACCAAGCCTAAAGCAGCAAAATAAGGCGTAGGATTAGAAGCAACAGCAATTAAACCCACAACCAGAGCTACCAATAA
CAAAAACATAAAATAAGTCATAATTCTTGCTCGGATTCTAACCGAGACCAGTGACTTGAAGAACCACCGTTGTAGTT
CAACTACAAGAACAATAATGGCAAGCCTACGAAAAACCCACCCACTAATAAAAATCGCCAACGACGCACTAGTTGAC
CTCCCCACACCATCTAACATTTCCGTATGATGAAATTTCGGGTCTCTCCTGGGGTTGTGTTTAATTACTCAAATCCT
AACCGGATTGTTCCTAGCCATACACTATACCTCTGACATCTCAACCGCATTTTCATCAGTAGTCCACATCTGCCGAG
ACGTGAACTATGGCTGACTTATCCGTAATATTCACGCCAACGGGGCATCATTCTTTTTTATCTGTATTTATATACAC
ATTGCCCGCGGCCTATACTATGGATCCTACCTGTATAAAGAAACCTGAAACATTGGGGTAGTCCTTCTCCTGTTAGT
TATAATAACAGCCTTTGTTGGCTACGTCCTTCCATGAGGACAAATATCCTTTTGAGGTGCCACAGTAATTACAAATC
TACTATCAGCAGTCCCCTACATAGGGGACACCCTCGTTCAATGAATCTGAGGTGGCTTCTCAGTAGACAACGCGACA
CTAACACGGATCTTCGCATTCCACTTCCTATTACCATTCATCGTCGCCGCCGCGACCATCCTACACCTACTTTTCCT
CCACGAAACAGGCTCGAACAACCCAGCCGGGCTAAACTCCGACGCAGACAAAATCTCCTTCCACCCATACTTCTCAT
ATAAAGACCTTCTAGGATTTGTAGTGATATTATTAGCCCTCACATCATTAGCACTATTCTCTCCAAATCTCCTGGGA
GACCCAGAGAATTTCACCCCTGCAAACCCACTAGTCACCCCTCCACATATCAAGCCAGAATGATACTTCCTATTTGC
TTACGCCATTCTACGGTCTATCCCGAACAAACTAGGAGGGGTCCTTGCATTGCTATTCTCTATTTTAGTCCTAATGG
TGGTACCAATTTTACACACCTCAAAACAACGAGGACTAACATTCCGCCCGATCACTCAATTCCTATTCTGAACCCTA
GTAGCAGATATAATTATCCTAACATGAATTGGAGGCATACCTGTAGAACATCCATACATTATCATCGGACAAATTGC
ATCAGTCCTTTACTTCGCATTATTCCTCATCCTCACTCCACTAGCAGGATGACTAGAAAATAAAGCACTAAAATGAG
CTTGCCCTAGTAGCTTAGTATTAAAGCATCGGTCTTGTAATCCGAAGATCGGAGGTTATAGTAACCTATATGGTTTA
ATACATATATATGTATTATATTACATAATGCATTAGTACAGATATATGTATTATCACCATTCATTTATATTAACCAT
AAAGCAAGTACTAACGTTCAAGACGTACATAAACCAAAATATTAAAACTCACAAATAATTTATTTAGACCCGGGAAA
TAGATTATTCCCCTATAATTGGCCCTCAAATATTTCCTTGAAATAATCAACTAGGATTTAACTCAACCATATTAATG
TAGTAAGAGACCACCAACTGGTTTATATTAAGGTATATCCTGCATGATAGGATCAGGGACACAAATTGTGGGGGTAA
CACAGAATGAACTATTACTTGCATCTGGCTTCAATCTCAGGAACATAACTGTAAAATTCCACCCTCGGATAATTATG
TCTGGCATATGGTTAAATGATGTGAGTACATACTCCTCATTAACCCCACATGCCGGGCATTCTTTTATATGCATAGG
GGTTCTCCTTTTTGGTTACCTTTCACCTTGCATCTCAGAGTGCAGGCTCAAATGACAAATCAAGGTTGAACATATTC
CTTGCTTGAATTCGAGTAGGTCAATTATTAAAAGACATAACTTAAGAATTACATACTTTTAACTCAAGTGCATAACA
CATTCATTCCTTCTTCAACTTACCCCTATATATATGCCCCCCCTCTCGGTTTCTGCGCGACAAACCCCCCTACCCCC
TACGCTCAGCAAATCCTGTTATCCTTGTCAAACCCCGAAACCAAGGAAGGTTCGAGAACGTATAAGCTAGCAAGTTG
ATTTATGGGCTAGCTATCCGCATTATATATATATATATACATACACATCGCATTAATTTGCCGCAAATTTCTCCAAA
TATTGGCCTAAAAGTCCCTATTAAATTTTTAGGGCGCGCCCCAATGCTAAAAAGTCCAACATTAAAAAGCGCTAGCG
TAGCTTAATACAAAGCATAACACTGAAGATGTTAAGATGGGCCCTGAGAAGCTCCGCATGCATAAAGGCATGGTCCC
GACCTTATTATCAGCTCTTACCCGACTTACACATGCAAGTCTCCGCAGTCCCGTGAGTATGCCCTCAATCCCCCGCC
CGGGGACGAGGAGCGGGCATCAGGCACAAATTTTAGCCCAAGACGCCTGGCCAAGCCACACCCCCAAGGGAATTCAG
CAGTGATAAACATTAAGCCATAAGTGAAAACTTGACTCAGTCAGGGCTAAGAGGGCCGGTAAAACT
SEQUENCE LISTING
<110>Yangtze River in China Three Gorges mandarin sturgeon research institute of group
<120>A kind of method and its application that amplification the Changjiang river fish chondriogen total order is combined based on degenerate primer
<130> 2011
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 1
arwtttaccg gcccthtta 19
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 2
agatagaaac tgacctggat t 21
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 3
gttcaacgat taaagtccta c 21
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 4
aarrgtagrt gratgtcgct g 21
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 5
gmaggcctyg atcctrcaaa 20
<210> 6
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 6
gcygcrtctt graakccta 19
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 7
gtttcaagyc arycrcataa 20
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 8
tragccgaaa tcarakgtc 19
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 9
agtayrwgtg acttccaatc a 21
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 10
catagctkyy rcttggaktt g 21
<210> 11
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 11
taggaaccaa aaactcttgg t 21
<210> 12
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 12
acaacggtgg ttyttcaag 19
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 13
tgacttgaar aaccaccgt 19
<210> 14
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 14
arwtttaccg gcccthtta 19
Claims (9)
1. the degenerate primer combination for expanding fish chondriogen total order, it is characterised in that including：
Pair of primers：
Mt-1F: 5’- ARWTTTACCGGCCCTHTTA-3’
Mt-1R: 5’-AGATAGAAACTGACCTGGATT-3’
Second pair of primer：
Mt-2F: 5’- GTTCAACGATTAAAGTCCTAC-3’
Mt-2R: 5’- AARRGTAGRTGRATGTCGCTG-3’
3rd pair of primer：
Mt-3F: 5’- GMAGGCCTYGATCCTRCAAA-3’
Mt-3R: 5’- GCYGCRTCTTGRAAKCCTA-3’
4th pair of primer：
Mt-4F: 5’- GTTTCAAGYCARYCRCATAA-3’
Mt-4R: 5’- TRAGCCGAAATCARAKGTC-3’
5th pair of primer：
Mt-5F: 5’- AGTAYRWGTGACTTCCAATCA-3’
Mt-5R: 5’- CATAGCTKYYRCTTGGAKTTG-3’
6th pair of primer：
Mt-6F: 5’- TAGGAACCAAAAACTCTTGGT-3’
Mt-6R: 5’- ACAACGGTGGTTYTTCAAG-3’
7th pair of primer：
Mt-7F: 5’- TGACTTGAARAACCACCGT-3’
Mt-7R: 5’- ARWTTTACCGGCCCTHTTA-3’
Wherein：Y represents base C or T；M represents base C or A；R represents base A or G；W represents base A or T；K represent base T or
G；H represents base A, T or C.
2. the TIANGEN for being used to expand fish mitochondria homologous segment combined containing degenerate primer described in claim 1
TIANamp Genomic DNA Kit（Blood/cell/tissue extracting genome DNA）Kit and reagent.
3. reagent according to claim 2, it is characterised in that anti-including one or more LA Taq archaeal dna polymerases, PCR
Answer buffer solution, dNTPs.
4. degenerate primer described in claim 1 combines the application in fingerling identification is carried out.
5. application according to claim 4, it is characterised in that comprise the following steps：
1）Extract the DNA in sample；
2）With step 1）The DNA of middle extraction is template, and pcr amplification reaction is carried out respectively using 7 pairs of primers in claim 1；
3）By step 2）7 obtained genetic fragments carry out gel extraction, connection conversion, sequencing splicing, obtain chondriogen
Complete sequence；
4）To step 3 in NCBI）Obtained sequence carries out Blast comparisons, determines species.
6. application according to claim 5, it is characterised in that pcr amplification reaction system is in every 25 μ l systems：
10xbuffer 2.5 μ l, MgCl2（25mM）1.5 μ l, dNTPs（10mM）1 μ l, concentration are that 100pmol/ μ l positive anti-primer is each
0.5 μ l, the μ l of template DNA 1, Taq enzyme（5U/μl）0.25 17.75 μ l of μ l, ddH2O.
7. application according to claim 5, it is characterised in that PCR reaction conditions are 94 DEG C of pre-degeneration 3min, and 94 DEG C are denatured
30s, 48-65 DEG C of annealing 30s, 72 DEG C of extension 2min, 30 circulations, 72 DEG C extend 10min, wherein, preferably 55 DEG C of annealing temperature.
8. application according to claim 5, it is characterised in that include the OMEGA glue reclaim kits for gel extraction
And for connecting conversion
pEASY-T1 Cloning Kit。
9. application according to claim 5, it is characterised in that 1）Linked system is in every 5 μ l systems：Glue reclaim product
1-4 μ l, pEASY-T1 Cloning Vector 1 μ l, remaining ddH2O is supplemented；2）Coupled reaction system is：25 DEG C of connections
20-30min；4 DEG C preserve for transformed clone sequencing；Wherein, linked system glue reclaim product preferably 1 μ l, reaction system is preferred
25 DEG C of connection 25min.
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| CN108913783A (en) * | 2018-07-12 | 2018-11-30 | 镇江华大检测有限公司 | For identifying molecular specificity labeled primers and its application of mandarin fish |
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